Compositions and methods for enhancing physiological performance and recovery time

ABSTRACT

Provided are methods for enhancing exercise (e.g., intense, eccentric, elevated temperature, repetitive, aerobic, and high altitude) performance, comprising administering electrokinetically-altered aqueous fluids comprising an ionic aqueous solution of stably configured charge-stabilized oxygen-containing nanostructures predominantly having an average diameter of less than 100 nanometers. In certain aspects, enhancing exercise performance comprises at least one of: reducing plasma inflammatory cytokines (e.g., IFN-alpha, ENA-78 and BDNF); ameliorating muscle/tendon damage or enhancing muscle/tendon recovery; reducing biomarkers of exercise-induced muscle injury (e.g., CK, plasma myoglobin); ameliorating exercise induced tendinosis, tendonitis, tenosynovitis, avulsion, and tendon strain associated with chronic repetitive movement or enhancing recovering therefrom; increasing VO 2  max; decreasing RPE; reducing blood lactate; preserving muscle contractile function (e.g., maximal force, joint ROM); reducing muscle soreness; ameliorating onset of fatigue in an exercising subject. Improved methods for producing electrokinetically altered aqueous fluids (including sports beverages) are also provided.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of U.S. patent application Ser. No.13/102,930, filed May 6, 2011 (issuing as U.S. Pat. No. 9,198,929 onDec. 1, 2015), which claims the benefit of U.S. Provisional ApplicationNo. 61/413,258, filed Nov. 12, 2010, U.S. Provisional Application No.61/358,798, filed Jun. 25, 2010, and U.S. Provisional Application No.61/332,669, filed May 7, 2010, the disclosures of which are herebyincorporated by reference herein in their entirety.

FIELD OF THE INVENTION

The present invention is directed generally to sports, exercise, energyand/or food beverages and more particularly toelectrokinetically-altered, oxygenated sports, energy and/or foodbeverages. Particular aspects relate to the use ofelectrokinetically-altered fluids (e.g., gas-enriched (e.g.,superoxygenated) electrokinetic fluids, comprising an ionic aqueoussolution of charge-stabilized oxygen-containing nanostructures)administered in an amount sufficient to provide for enhancing at leastone of physiological performance and recovery time. Certain aspectsrelate to administering said beverages in an amount sufficient toprovide for preventing exercise-induced muscle and/or tendon damageand/or enhancing/facilitating muscle and/or tendon recovery fromexercise and/or exercise-induced damage). Certain aspects relate topreventing and/or ameliorating and/or enhancing recovery from muscle ortendon strain associated with chronic, repetitive movement. Furtheraspects relate to ameliorating the effects of physical exertion of thesubject. Certain aspects relate to improved methods for producingelectrokinetically altered aqueous fluids (including sports beverages).

BACKGROUND OF THE INVENTION

In humans and other animals, exercise, strenuous training, and exposureto elements (e.g., sunlight, wind, rain, cold and heat) can result insignificant physiological changes. Subjects exercising or training areat risk for developing injuries (e.g., muscle and/or tendon damage),especially subjects doing so in extreme conditions (e.g., cold or heat,high altitude, long durations, high intensity, repetitive, aerobic,contact sports, etc.). For example, excessive heat temperatures, inparticular, environmental heat illnesses include but are not limited toheat syncope, heat exhaustion, dehydration syndrome, and heat stroke.The potentially fatal clinical syndrome of heat stroke has beendescribed in marathon runners, military recruits, football players, andin hot industrial environments. An epidemic appearance of heat strokehas been described during heat waves in urban areas (Ferguson, M., andM. M. O'brien, “Heat Stroke In New York City: Experience With 25 Cases,”N.Y. State J. Med. 60:2531-2538, 1960).

Likewise, impaired or incomplete recovery following high-intensityexercise can negatively affect physical performance and delay functionalprogression, thereby reducing an athlete's chance of performing at hisor her peak level. Athletes are constantly seeking ways to preventexercise-induced muscle damage and facilitate muscle recovery fromstrenuous exercise. For example, athletes have used dietary supplementsextensively to facilitate tissue growth and repair followingmuscle-damaging events such as high-intensity resistance exercise andparticipation in contact sports. Following strenuous exercise, an acuteinflammatory response drives the repair process by synthesizing andreleasing chemical mediators locally in the injured muscle. However,while inflammatory mediators may help attract growth factors used forprotein synthesis and muscle repair, excessive inflammatory response maydamage muscle and thereby hinder the repair process.

“Dehydration syndrome” may be characterized and/or accompanied by lossof appetite and limited capacity for work. Evidence of heat exhaustionbecomes apparent with losses of, for example, 5% of the body water, andat about 7% loss of body water disorientation and hallucinations mayoccur. Losses of body water of 10% or greater are extremely hazardousand lead to heat stroke and death if not treated immediately. Heatstroke is accompanied by high body temperature (41.1° C.-43.3° C.; 106°F.-110° F.), deep coma, and in most cases a complete absence ofsweating, and failure of major organ systems.

At least three factors determine the thermal balance of the body:metabolic heat production, heat exchange between the organism and itssurroundings, and heat loss by the evaporation of sweat (Knochel, J. P.,[1980] “Clinical Physiology Of Heat Exposure,” In Clinical Disorders OfFluid And Electrolyte Metabolism, M. H. Maxwell and C. R. Kleeman, Eds.,Mcgraw-Hill, New York). For the subject exercising or working,particularly in a hot environment, the capacity to dissipatemetabolically produced heat depends for the most part on the subject'sability to form and vaporize sweat (Costill, D. L., and K. E. Sparks,“Rapid Fluid Replacement Following Thermal Dehydration,” J. Appl.Physiol. 34(3):299-303, 1973; Greenleaf, J. E., “Hyperthermia AndExercise,” Int. Rev. Physiol. 20:157-208, 1979).

During exercise, especially in a hot environment, serious deficits ineffective circulating volume may occur. Muscular work, independent ofenvironment, results in massive shunting of blood to skeletal muscle,along with a substantial loss of plasma volume into the working muscle.Moreover, effective circulating volume is also diminished by losses ofsweat (Knochel [1980] supra). The deficit in intravascular volumeimpedes the delivery of heated blood to the periphery for evaporativecooling. Thus, in the dehydrated exercising subject, there is aprogressive increase in the core body temperature as sweat lossesaccumulate.

Notable among the many physiological responses to physical exertion areincreased body temperature, perspiration and pulse rate, a decrease inthe blood volume, and biochemical changes associated with the metabolismof compounds to produce energy. In addition, approximately 90% of thebody's energy is created by oxygen. All of the activities of the body,from brain function to elimination, are regulated by oxygen. Bloodplasma holds approximately three percent (3%) dissolved oxygen, and redblood cells (hemoglobin) hold ninety-seven percent (97%). From the redblood cells the oxygen passes out into the plasma and is transferred tocells that need oxygen during metabolic processes. These cells pass CO₂back to the plasma where it is then picked up by the red blood cells.This process rapidly increases, for example, during exercise andstrenuous training.

Prior research in the art has focused on the ability of glycerol tocause water retention. However, water retention alone has little or nocorrelation with enhanced endurance or physiological performance. Inorder to have a beneficial effect on endurance and performance, watermust be appropriately allocated throughout the body. Mere reduction inurine output is insufficient. Water must be available for sweating(efficient cooling), hydration of cells, and plasma volume must bemaintained. Only if these physiological objectives are met can enduranceand performance be enhanced.

Osmotic pressure is primarily responsible for the direction and rate ofmovement of water across semi permeable membranes in the body. Thus,water will move across a semi permeable membrane such that the net flowof water will be across the membrane into the fluid which initially hadthe highest concentration of solutes, and thus the allocation of waterbetween digestive organs, blood plasma, and cells depends upon therelative osmotic pressures between these sites. Although it has beenestablished that the ingestion of massive amounts of glycerol results inthe retention of water within the body (i.e., the rate of urine flow isdecreased), this observation alone produces no information as to whetherthe body's physiological responses to heat or physical exertion havebeen enhanced. For example, a large concentration of glycerol in thestomach or intestine can cause water to move across the gastrointestinalmembranes into the digestive tract and cause detrimental responses tophysical exertion and heat exposure. Alternatively, high concentrationsof plasma glycerol can cause water to leave the cells and enter theplasma, resulting in detrimental cellular dehydration.

There is, therefore, a pronounced need in the art for novel andeffective methods to enhance exercise performance and recovery time andrelated conditions as described herein.

BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 is a partial cross-section, partial block diagram of a prior artmixing device.

FIG. 2 is block diagram of an exemplary embodiment of a mixing device.

FIG. 3 is an illustration of an exemplary system for delivering a firstmaterial to the mixing device of FIG. 2.

FIG. 4 is a fragmentary partial cross-sectional view of a top portion ofthe mixing device of FIG. 2.

FIG. 5 is a fragmentary cross-sectional view of a first side portion ofthe mixing device of FIG. 2.

FIG. 6 is a fragmentary cross-sectional view of a second side portion ofthe mixing device of FIG. 2.

FIG. 7 is a fragmentary cross-sectional view of a side portion of themixing device of FIG. 2 located between the first side portion of FIG. 5and the second side portion of FIG. 6.

FIG. 8 is a perspective view of a rotor and a stator of the mixingdevice of FIG. 2.

FIG. 9 is a perspective view of an inside of a first chamber of themixing device of FIG. 2.

FIG. 10 is a fragmentary cross-sectional view of the inside of a firstchamber of the mixing device of FIG. 2 including an alternate embodimentof the pump 410.

FIG. 11 is a perspective view of an inside of a second chamber of themixing device of FIG. 2.

FIG. 12 is a fragmentary cross-sectional view of a side portion of analternate embodiment of the mixing device.

FIG. 13 is a perspective view of an alternate embodiment of a centralsection of the housing for use with an alternate embodiment of themixing device.

FIG. 14 is a fragmentary cross-sectional view of an alternate embodimentof a bearing housing for use with an alternate embodiment of the mixingdevice.

FIG. 15 is a cross-sectional view of the mixing chamber of the mixingdevice of FIG. 2 taken through a plane orthogonal to the axis ofrotation depicting a rotary flow pattern caused by cavitation bubbleswhen a through-hole of the rotor approaches (but is not aligned with) anaperture of the stator.

FIG. 16 is a cross-sectional view of the mixing chamber of the mixingdevice of FIG. 2 taken through a plane orthogonal to the axis ofrotation depicting a rotary flow pattern caused by cavitation bubbleswhen the through-hole of the rotor is aligned with the aperture of thestator.

FIG. 17 is a cross-sectional view of the mixing chamber of the mixingdevice of FIG. 2 taken through a plane orthogonal to the axis ofrotation depicting a rotary flow pattern caused by cavitation bubbleswhen a through-hole of the rotor that was previously aligned with theaperture of the stator is no longer aligned therewith.

FIG. 18 is a side view of an alternate embodiment of a rotor.

FIG. 19 is an enlarged fragmentary cross-sectional view taken through aplane orthogonal to an axis of rotation of the rotor depicting analternate configuration of through-holes formed in the rotor andthrough-holes formed in the stator.

FIG. 20 is an enlarged fragmentary cross-sectional view taken through aplane passing through and extending along the axis of rotation of therotor depicting a configuration of through-holes formed in the rotor andthrough-holes formed in the stator.

FIG. 21 is an enlarged fragmentary cross-sectional view taken through aplane passing through and extending along the axis of rotation of therotor depicting an alternate offset configuration of through-holesformed in the rotor and through-holes formed in the stator.

FIG. 22 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 23 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 24 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 25 is an illustration of a shape that may be used to construct thethrough-holes of the rotor and/or the apertures of the stator.

FIG. 26 is an illustration of an electrical double layer (“EDL”) formednear a surface.

FIG. 27 is a perspective view of a model of the inside of the mixingchamber.

FIG. 28 is a cross-sectional view of the model of FIG. 27.

FIG. 29 is an illustration of an experimental setup.

FIG. 30 illustrates dissolved oxygen levels in water processed withoxygen in the mixing device of FIG. 2 and stored a 500 ml thin walledplastic bottle and a 1,000 ml glass bottle each capped at 65°Fahrenheit.

FIG. 31 illustrates dissolved oxygen levels in water processed withoxygen in the mixing device of FIG. 2 and stored in a 500 ml plasticthin walled bottle and a 1,000 ml glass bottle both refrigerated at 39°Fahrenheit.

FIG. 32 illustrates the dissolved oxygen levels in GATORADE® processedwith oxygen in the mixing device of FIG. 2 and stored in 32 oz.GATORADE® bottles having an average temperature of 55° Fahrenheit.

FIG. 33 illustrates the dissolved oxygen retention of a 500 ml braunbalanced salt solution processed with oxygen in the mixing device ofFIG. 2.

FIG. 34 illustrates a further experiment wherein the mixing device ofFIG. 2 is used to sparge oxygen from water by processing the water withnitrogen in the mixing device of FIG. 2.

FIG. 35 illustrates the sparging of oxygen from water by the mixingdevice of FIG. 2 at standard temperature and pressure.

FIG. 36 is an illustration of a nanocage.

FIG. 37 illustrates the Rayleigh scattering effects produced by a sampleof the water processed with oxygen by the mixing device of FIG. 2.

FIGS. 38-41 illustrate the inventive oxygen-enriched fluid testedpositive for reactivity with horseradish peroxidase by pyrogallol, whilethe pressure pot and fine bubbled water samples had far less reactivity.

FIG. 42 illustrates pyrogallol/HRP assays as described herein, showingthat oxygen is required for the reaction with pyrogallol in the presenceof horseradish peroxidase, as inventive fluid enriched with other gases(argon and nitrogen) did not react in the same manner.

FIG. 43 illustrates the hydrogen peroxide positive control showed astrong reactivity, while none of the other fluids tested reacted withthe glutathione.

FIG. 44 illustrates T7 DNA shows a conformational change at about 50° C.in the control (deionized water), whereas the DNA in the oxygen-enrichedinventive fluid remains intact until about 60° C.

FIGS. 45A and 45B illustrate a graphical representation of a exemplaryembodiments of a bioreactor system 3300 a.

FIG. 46 shows detailed portions of exemplary embodiments of thebioreactor system 3300 a of FIGS. 45A and 45B.

FIG. 47A shows the effect that RNS60 has on DEP mediated IL-8stimulation in human bronchial epithelial cells.

FIG. 47B shows that RNS60 regulates rTNFa induced IL-8, and it retainsits biological activity at room temperature for days.

FIG. 48 illustrates the benefits of consuming theelectrokinetically-altered fluids for exercising individuals. Theresults (see Table 4 herein below) indicate that the beverage had aneffect on all 3 measured parameters of exercise performance, and thatthe direction of the effect was favorable in all 3 areas (i.e., positivefor VO₂ max, negative for RPE (rating of perceived exertion), andnegative for lactate).

FIG. 49 shows a study design overview.

FIGS. 50A and 50B show that RB consumption improves VO₂max in fitterathletes.

FIG. 51 shows that RB consumption decreases RPE.

FIG. 52 shows time point differences in levels of plasma myoglobin.

FIG. 53 shows time point differences in plasma CK levels.

FIGS. 54A and 54B shows that RB inhibited the exercise-induced increaseof plasma levels of IFN-α (A) and ENA-78 (B).

FIG. 55 shows that RB consumption prevents the rise in BDNF plasmaconcentration 24 hours after the exercise trial.

FIG. 56 shows the effect of RB consumption on circulating sCD40L levels.

SUMMARY OF EXEMPLARY EMBODIMENTS

Particular aspects provide methods for enhancing exercise performance,comprising administering to a subject in need thereof, anelectrokinetically altered aqueous fluid comprising an ionic aqueoussolution of charge-stabilized oxygen-containing nanostructurespredominantly having an average diameter of less than 100 nanometers andstably configured in the ionic aqueous solution in an amount sufficientto provide for enhancing at least one of exercise performance andrecovery time, wherein a method for enhancing exercise performance isafforded.

In certain method aspects, enhancing exercise performance comprisesreducing exercise-induced increases of plasma inflammatory cytokinelevels in the subject. In particular aspects, the exercise-inducedplasma inflammatory cytokine is one selected from the group consistingof interferon-alpha (IFN-alpha), epithelial neutrophil activatingprotein 78 (ENA-78), and brain-derived neurotrophic factor (BDNF).

In particular method aspects, enhancing exercise performance comprisesat least one of preventing or ameliorating exercise-mediated muscleand/or tendon damage and enhancing muscle and/or tendon recoverytherefrom (e.g., comprises at least one of preventing or alleviatingextent of muscle fiber micro-injury, and enhancing recovery therefrom;comprises reducing biomarkers of exercise-induced muscle injury (e.g.,creatine kinase (CK), plasma myoglobin); or comprises ameliorating orenhancing recovering from at least one of exercise induced tendinosis,tendonitis, tenosynovitis, avulsion, and tendon strain associated withchronic, repetitive movement). In certain aspects, enhancing exerciseperformance comprises at least one of: increasing the maximum amount ofoxygen that the subject can utilize during intense or maximal exercise(VO₂ max); decreasing the rating of perceived exertion (RPE); reducingexercise-mediated increase blood lactate levels; preserving musclecontractile function, preferably maximal force or joint ROM; reducingmuscle soreness; and ameliorating the onset of fatigue in response toexercise in the subject.

In particular method aspects, the exercise comprises at least one ofintense exercise, eccentric exercise, exercise in elevated ambienttemperature, repetitive exercise, aerobic exercise, and high altitudeexercise.

In certain aspects, the electrokinetically altered aqueous fluid issuperoxygenated (e.g., wherein, the electrokinetically-altered aqueousfluid comprises oxygen in an amount of at least 15, ppm, at least 25ppm, at least 30 ppm, at least 40 ppm, at least 50 ppm, or at least 60ppm oxygen at atmospheric pressure)

In certain aspects, the ionic aqueous solution comprises a salinesolution, and may comprise at least one ion or salt disclosed in Tables1 an 2 herein.

In particular aspects, the charge-stabilized oxygen-containingnanostructures are stably configured in the ionic aqueous fluid in anamount sufficient to provide, upon contact of a living cell by thefluid, modulation of at least one of cellular membrane potential andcellular membrane conductivity. In particular aspects, the ability tomodulate of at least one of cellular membrane potential and cellularmembrane conductivity persists for at least two, at least three, atleast four, at least five, at least 6, at least 12 months, or longerperiods, in a closed gas-tight container, optimally at about 4° C. Incertain aspects, modulation of at least one of cellular membranepotential and cellular membrane conductivity comprises altering of aconformation, ligand binding activity, or a catalytic activity of amembrane associated protein. In certain aspects, modulation of at leastone of cellular membrane potential and cellular membrane conductivity,comprises modulating whole-cell conductance. In particular aspects,modulation of at least one of cellular membrane potential and cellularmembrane conductivity comprises modulation of at least one of: a calciumdependant cellular messaging pathway or system; phospholipase Cactivity; and adenylate cyclase (AC) activity.

In particular embodiments, administering the electrokinetically alteredaqueous fluid comprises oral administration of an aqueous solution orsports beverage (e.g., a sports beverage comprising a sugar,carbohydrate, electrolyte or other sports beverage ingredient).

Additional aspects provide improved methods for producing anelectrokinetically-altered oxygenated aqueous fluid or solution,comprising: providing a flow of aqueous fluid material between twospaced surfaces defining a mixing volume therebetween; and introducingoxygen gas into the flowing aqueous fluid material, at or substantiallyat the temperature of highest density of the aqueous fluid material,within the mixing volume under conditions suitable to infuse at least 20ppm gas into the material in less than 400 milliseconds, wherein anelectrokinetically altered aqueous fluid comprising an ionic aqueoussolution of charge-stabilized oxygen-containing nanostructurespredominantly having an average diameter of less than 100 nanometers andstably configured in the ionic aqueous fluid is provided. In particularaspects, the dwell time of the flowing material within the mixing volumeis greater than 0.06 seconds or greater than 0.1 seconds. In certainaspects, the ratio of surface area to the volume is at least 12, atleast 20, at least 30, at least 40, or at least 50.

Yet additional aspects provide improved methods for producing anelectrokinetically-altered oxygenated aqueous fluid or solution,comprising use of a mixing device for creating an output mixture bymixing a first aqueous material and a second material, the devicecomprising: a first chamber configured to receive the first aqueousmaterial from a source of the first aqueous material; a stator; a rotorhaving an axis of rotation, the rotor being disposed inside the statorand configured to rotate about the axis of rotation therein, at leastone of the rotor and stator having a plurality of through-holes; amixing chamber defined between the rotor and the stator, the mixingchamber being in fluid communication with the first chamber andconfigured to receive the first aqueous material therefrom, and oxygenbeing provided to the mixing chamber via the plurality of through-holesformed in the one of the rotor and stator; a second chamber in fluidcommunication with the mixing chamber and configured to receive theoutput material therefrom; and a first internal pump housed inside thefirst chamber, the first internal pump being configured to pump thefirst aqueous material from the first chamber into the mixing chamber,at or substantially at the temperature of highest density of the aqueousfluid material, wherein an electrokinetically altered aqueous fluidcomprising an ionic aqueous solution of charge-stabilizedoxygen-containing nanostructures predominantly having an averagediameter of less than 100 nanometers and stably configured in the ionicaqueous fluid is provided.

Further aspects provide improved methods for producing anelectrokinetically-altered oxygenated aqueous fluid or solution,comprising use of a mixing device for creating an output mixture bymixing a first aqueous material and a second material, the devicecomprising: a stator; a rotor having an axis of rotation, the rotorbeing disposed inside the stator and configured to rotate about the axisof rotation therein; a mixing chamber defined between the rotor and thestator, the mixing chamber having an open first end through which thefirst aqueous material enters the mixing chamber at or substantially atthe temperature of highest density of the aqueous fluid material, and anopen second end through which the output material exits the mixingchamber, the second material, oxygen gas, entering the mixing chamberthrough at least one of the rotor and the stator; a first chamber incommunication with at least a majority portion of the open first end ofthe mixing chamber; and a second chamber in communication with the opensecond end of the mixing chamber, to electrokinetically alter theaqueous material, wherein an electrokinetically altered aqueous fluidcomprising an ionic aqueous solution of charge-stabilizedoxygen-containing nanostructures predominantly having an averagediameter of less than 100 nanometers and stably configured in the ionicaqueous fluid is provided.

In particular aspects of the methods, the first internal pump isconfigured to impart a circumferential velocity into the aqueousmaterial before it enters the mixing chamber.

Yet further aspects provide methods for producing anelectrokinetically-altered oxygenated aqueous fluid or solution in anarcuate mixing chamber formed between two contoured surfaces to createan output mixture, the arcuate mixing chamber having a first end portionopposite a second end portion, the method comprising: providing a firstaqueous material; introducing the first aqueous material into the firstend portion of the arcuate mixing chamber, at or substantially at thetemperature of highest density of the aqueous fluid material, in a flowdirection having a first component that is substantially tangent to thearcuate mixing chamber and a second component that is directed towardthe second end portion; and introducing oxygen gas into the arcuatemixing chamber though at least one of the two contoured surfaces betweenthe first end portion of the arcuate mixing chamber and the second endportion of the arcuate mixing chamber, wherein an electrokineticallyaltered aqueous fluid comprising an ionic aqueous solution ofcharge-stabilized oxygen-containing nanostructures predominantly havingan average diameter of less than 100 nanometers and stably configured inthe ionic aqueous fluid is provided. In particular embodiments, thefirst end portion of the mixing chamber is coupled to a first chamber,the method further comprising: before introducing the first aqueousmaterial into the first end portion of the arcuate mixing chamber,introducing the first aqueous material into the first chamber, andimparting a circumferential flow into said material in the firstchamber. In further embodiments, the first end portion of the mixingchamber is coupled to a first chamber, the mixing chamber is formedbetween an outer contoured surface of a rotating cylindrical rotor andan inner contoured surface of a stationary cylindrical stator, and therotor rotates inside the stator about an axis of rotation, the methodfurther comprising: before introducing the first aqueous material intothe first end portion of the arcuate mixing chamber, introducing thefirst aqueous material into the first chamber, and imparting acircumferential flow substantially about an axis of rotation into saidmaterial in the first chamber; introducing oxygen gas into a hollowportion of a rotating rotor having a plurality of through-holes, eachthrough-hole of the plurality extending from the hollow portion to theouter contoured surface of the rotor; flowing the oxygen gas from thehollow portion of the rotating rotor through the plurality ofthrough-holes into the mixing chamber; flowing the aqueous material fromthe first chamber into the mixing chamber; and rotating the rotorrelative to the stator thereby mixing aqueous material and the oxygengas together inside the mixing chamber.

In further aspects of all of the above methods, the aqueous fluidmaterial comprises at least one salt or ion from Tables 1 and 2disclosed herein.

In certain embodiments, the methods comprises production of a sports orexercise beverage, or of a component thereof.

DETAILED DESCRIPTION OF THE INVENTION

Provided are methods for enhancing exercise performance, comprisingadministering electrokinetically altered aqueous fluids comprising anionic aqueous solution of charge-stabilized oxygen-containingnanostructures predominantly having an average diameter of less than 100nanometers and stably configured in the ionic aqueous solution. Incertain aspects, enhancing exercise performance comprises: reducingplasma inflammatory cytokines (e.g., IFN-alpha, ENA-78 and BDNF); and/orameliorating exercise-mediated muscle/tendon damage and enhancingmuscle/tendon recovery; and/or reducing biomarkers of exercise-inducedmuscle injury (e.g., CK, plasma myoglobin); and/or ameliorating orenhancing recovering from exercise induced tendinosis, tendonitis,tenosynovitis, avulsion, and tendon strain associated with chronicrepetitive movement; and/or increasing VO₂ max; and/or decreasing RPE;reducing blood lactate levels; and/or preserving muscle contractilefunction (e.g., maximal force or joint ROM); and/or reducing musclesoreness; and/or ameliorating the onset of fatigue in response toexercise in the subject. In particular method aspects, the exercisecomprises at least one of intense exercise, eccentric exercise, exercisein elevated ambient temperature, repetitive exercise, aerobic exercise,and high altitude exercise. Improved methods for producingelectrokinetically altered aqueous fluids (including sports beverages)are also provided.

Provided are electrokinetically-altered fluid sports beveragecompositions, comprising an electrokinetically-altered aqueous fluidcomprising an ionic aqueous solution of charge-stabilizedoxygen-containing nanostructures substantially having an averagediameter of less than about 100 nanometers and stably configured in theionic aqueous fluid in an amount sufficient to provide for enhancing atleast one of physiological performance and recovery time. In certainaspects, the charge-stabilized oxygen-containing nanostructures arestably configured in the ionic aqueous fluid in an amount sufficient toprovide, upon contact of a living cell by the fluid, modulation of atleast one of cellular membrane potential and cellular membraneconductivity. In certain aspects, the charge-stabilizedoxygen-containing nanostructures are the major charge-stabilizedgas-containing nanostructure species in the fluid. In particularaspects, the percentage of dissolved oxygen molecules present in thefluid as the charge-stabilized oxygen-containing nanostructures is apercentage selected from the group consisting of greater than: 0.01%,0.1%, 1%, 5%; 10%; 15%; 20%; 25%; 30%; 35%; 40%; 45%; 50%; 55%; 60%;65%; 70%; 75%; 80%; 85%; 90%; and 95%. In certain embodiments, the totaldissolved gas is substantially present in the charge-stabilizedgas-containing nanostructures. In certain aspects, the charge-stabilizedgas-containing nanostructures substantially have an average diameter ofless than a size selected from the group consisting of: 90 nm; 80 nm; 70nm; 60 nm; 50 nm; 40 nm; 30 nm; 20 nm; 10 nm; and less than 5 nm.

In particular aspects, the ionic aqueous solution comprises a salinesolution. In certain aspects, the fluid is super-oxygenated.

In certain embodiments, the charge-stabilized gas-containingnanostructures comprise at least one ion or salt disclosed in Tables 1an 2 herein, or at least one ion selected from the group consisting ofalkali metal based salts including Li+, Na+, K+, Rb+, and Cs+, alkalineearth based salts including Mg++ and Ca++, and transition metal-basedpositive ions including Cr, Fe, Co, Ni, Cu, and Zn, in each case alongwith any suitable counterionic components.

In certain aspects, the fluid comprises at least one of a form ofsolvated electrons, and an electrokinetically modified or charged oxygenspecies. In certain aspects, the form of solvated electrons orelectrokinetically modified or charged oxygen species are present in anamount of at least 0.01 ppm, at least 0.1 ppm, at least 0.5 ppm, atleast 1 ppm, at least 3 ppm, at least 5 ppm, at least 7 ppm, at least 10ppm, at least 15 ppm, or at least 20 ppm. In certain aspects, theelectrokinetically-altered fluid comprises a form of solvated electronsstabilized, at least in part, by molecular oxygen.

In particular aspects, the ability to modulate of at least one ofcellular membrane potential and cellular membrane conductivity persistsfor at least two, at least three, at least four, at least five, at least6, at least 12 months, or longer periods, in a closed gas-tightcontainer, optimally at about 4° C.

In certain aspects, alteration of the electrokinetically altered aqueousfluid comprises exposure of the fluid to hydrodynamically-induced,localized electrokinetic effects. In certain aspects, exposure to thelocalized electrokinetic effects comprises exposure to at least one ofvoltage pulses and current pulses. In certain aspects, the exposure ofthe fluid to hydrodynamically-induced, localized electrokinetic effects,comprises exposure of the fluid to electrokinetic effect-inducingstructural features of a device used to generate the fluid.

In certain embodiments, modulation of at least one of cellular membranepotential and cellular membrane conductivity comprises altering of aconformation, ligand binding activity, or a catalytic activity of amembrane associated protein. In certain aspects, the membrane associatedprotein comprises at least one selected from the group consisting ofreceptors, transmembrane receptors, ion channel proteins, intracellularattachment proteins, cellular adhesion proteins, integrins, etc. Incertain aspects, the transmembrane receptor comprises a G-ProteinCoupled Receptor (GPCR). In particular aspects, the G-Protein CoupledReceptor (GPCR) interacts with a G protein a subunit. In certainaspects, the G protein a subunit comprises at least one selected fromthe group consisting of Gα_(s), Gα_(i), Gα_(q), and Gα₁₂ (e.g., whereinthe G protein α subunit is Gα_(q)).

In certain aspects, modulation of at least one of cellular membranepotential and cellular membrane conductivity, comprises modulatingwhole-cell conductance. In particular aspects, modulating whole-cellconductance, comprises modulating at least one of a linear or non-linearvoltage-dependent contribution of the whole-cell conductance.

In certain embodiments, modulation of at least one of cellular membranepotential and cellular membrane conductivity comprises modulation of acalcium dependant cellular messaging pathway or system. In certainaspects, modulation of at least one of cellular membrane potential andcellular membrane conductivity comprises modulation of phospholipase Cactivity. In certain aspects, modulation of at least one of cellularmembrane potential and cellular membrane conductivity comprisesmodulation of adenylate cyclase (AC) activity.

In certain aspects, the electrokinetically-altered sports beveragecomposition comprises dissolved oxygen in an amount of at least 8 ppm,at least 15, ppm, at least 25 ppm, at least 30 ppm, at least 40 ppm, atleast 50 ppm, or at least 60 ppm oxygen at atmospheric pressure. Incertain aspects, the gas in the charge-stabilized nanostructures of thefluid or solution is present in an amount of at least 25 ppm.

Additionally provided are methods for producing a sports beveragecomposition, comprising: providing a sports beverage fluid formulationor composition; providing a flow of the sports beverage fluidformulation or composition material between two spaced surfaces inrelative motion and defining a mixing volume therebetween, wherein thedwell time of a single pass of the flowing fluid material within andthrough the mixing volume is greater than 0.06 seconds or greater than0.1 seconds; and introducing oxygen gas into the flowing fluid materialwithin the mixing volume under conditions suitable to dissolve at least20 ppm, at least 25 ppm, at least 30, at least 40, at least 50, or atleast 60 ppm gas into the material, and electrokinetically alter thefluid material, wherein a sports beverage composition comprising anelectrokinetically altered aqueous fluid comprising an ionic aqueoussolution of charge-stabilized gas-containing nanostructuressubstantially having an average diameter of less than about 100nanometers and stably configured in the ionic aqueous fluid in an amountsufficient to provide for enhancing at least one of physiologicalperformance and recovery time is provided. In certain aspects, theoxygen gas is infused into the material in less than 100 milliseconds,less than 200 milliseconds, less than 300 milliseconds, or less than 400milliseconds.

Further aspects provide a method of producing a sports beveragecomposition, comprising: providing a sports beverage fluid formulationor composition; providing a flow of the sports beverage fluidformulation or composition material between two spaced surfaces defininga mixing volume therebetween; and introducing oxygen gas into theflowing material within the mixing volume under conditions suitable toinfuse at least 20 ppm, at least 25 ppm, at least 30, at least 40, atleast 50, or at least 60 ppm gas into the material in less than 100milliseconds, less than 200 milliseconds, less than 300 milliseconds, orless than 400 milliseconds, to electrokinetically alter the sportsbeverage fluid formulation or composition, wherein a sports beveragefluid formulation or composition comprising an electrokineticallyaltered aqueous fluid comprising an ionic aqueous solution ofcharge-stabilized gas-containing nanostructures substantially having anaverage diameter of less than about 100 nanometers and stably configuredin the ionic aqueous fluid in an amount sufficient to provide forenhancing at least one of physiological performance and recovery time isprovided. In certain aspects, the dwell time of the flowing materialwithin the mixing volume is greater than 0.06 seconds or greater than0.1 seconds. In certain aspects, the ratio of surface area to the volumeis at least 12, at least 20, at least 30, at least 40, or at least 50.

Yet further aspects provide a method of producing a sports beveragecomposition, comprising use of a mixing device for creating an outputmixture by mixing a first material and a second material, the devicecomprising: providing a sports beverage fluid formulation orcomposition; a first chamber configured to receive the sports beveragefluid formulation or composition material from a source of the firstmaterial; a stator; a rotor having an axis of rotation, the rotor beingdisposed inside the stator and configured to rotate about the axis ofrotation therein, at least one of the rotor and stator having aplurality of through-holes; a mixing chamber defined between the rotorand the stator, the mixing chamber being in fluid communication with thefirst chamber and configured to receive the sports beverage fluidformulation or composition material therefrom, and oxygen being providedto the mixing chamber via the plurality of through-holes formed in theone of the rotor and stator; a second chamber in fluid communicationwith the mixing chamber and configured to receive the output materialtherefrom; and a first internal pump housed inside the first chamber,the first internal pump being configured to pump the sports beveragefluid formulation or composition material from the first chamber intothe mixing chamber, to electrokinetically alter the sports beveragefluid formulation or composition material, wherein a sports beveragefluid formulation or composition comprising an electrokineticallyaltered aqueous fluid comprising an ionic aqueous solution ofcharge-stabilized gas-containing nanostructures substantially having anaverage diameter of less than about 100 nanometers and stably configuredin the ionic aqueous fluid in an amount sufficient to provide forenhancing at least one of physiological performance and recovery time isprovided.

Additional aspects provide a method of producing a sports beveragecomposition, comprising use of a mixing device for creating an outputmixture by mixing a first material and a second material, the devicecomprising: providing a sports beverage fluid formulation orcomposition; a stator; a rotor having an axis of rotation, the rotorbeing disposed inside the stator and configured to rotate about the axisof rotation therein; a mixing chamber defined between the rotor and thestator, the mixing chamber having an open first end through which thesports beverage fluid formulation or composition material enters themixing chamber and an open second end through which the output materialexits the mixing chamber, the second material, oxygen gas, entering themixing chamber through at least one of the rotor and the stator; a firstchamber in communication with at least a majority portion of the openfirst end of the mixing chamber; and a second chamber in communicationwith the open second end of the mixing chamber, to electrokineticallyalter the sports beverage fluid formulation or composition material,wherein a sports beverage fluid formulation or composition materialcomprising an electrokinetically altered aqueous fluid comprising anionic aqueous solution of charge-stabilized gas-containingnanostructures substantially having an average diameter of less thanabout 100 nanometers and stably configured in the ionic aqueous fluid inan amount sufficient to provide for enhancing at least one ofphysiological performance and recovery time is provided. In certainaspects, the first internal pump is configured to impart acircumferential velocity into the sports beverage fluid formulation orcomposition material before it enters the mixing chamber

Yet additional aspects provide a method of producing a sports beveragecomposition material in an arcuate mixing chamber formed between twocontoured surfaces to create an output mixture, the arcuate mixingchamber having a first end portion opposite a second end portion, themethod comprising: providing a sports beverage fluid formulation orcomposition; introducing the sports beverage fluid formulation orcomposition material into the first end portion of the arcuate mixingchamber in a flow direction having a first component that issubstantially tangent to the arcuate mixing chamber and a secondcomponent that is directed toward the second end portion; andintroducing oxygen gas into the arcuate mixing chamber though at leastone of the two contoured surfaces between the first end portion of thearcuate mixing chamber and the second end portion of the arcuate mixingchamber, wherein a sports beverage fluid formulation or compositionmaterial comprising an electrokinetically-altered aqueous fluidcomprising an ionic aqueous solution of charge-stabilized gas-containingnanostructures substantially having an average diameter of less thanabout 100 nanometers and stably configured in the ionic aqueous fluid inan amount sufficient to provide for enhancing at least one ofphysiological performance and recovery time is provided. In certainaspects, the first end portion of the mixing chamber is coupled to afirst chamber, the method further comprising: before introducing thesports beverage fluid formulation or composition material into the firstend portion of the arcuate mixing chamber, introducing the sportsbeverage fluid formulation or composition material into the firstchamber, and imparting a circumferential flow into said material in thefirst chamber. In certain aspects, the first end portion of the mixingchamber is coupled to a first chamber, the mixing chamber is formedbetween an outer contoured surface of a rotating cylindrical rotor andan inner contoured surface of a stationary cylindrical stator, and therotor rotates inside the stator about an axis of rotation, the methodfurther comprising: before introducing the sports beverage fluidformulation or composition material into the first end portion of thearcuate mixing chamber, introducing the sports beverage fluidformulation or composition material into the first chamber, andimparting a circumferential flow substantially about an axis of rotationinto said material in the first chamber; introducing oxygen has into ahollow portion of a rotating rotor having a plurality of through-holes,each through-hole of the plurality extending from the hollow portion tothe outer contoured surface of the rotor; flowing the oxygen gas fromthe hollow portion of the rotating rotor through the plurality ofthrough-holes into the mixing chamber; flowing the sports beverage fluidformulation or composition material from the first chamber into themixing chamber; and rotating the rotor relative to the stator therebymixing the sports beverage fluid formulation or composition material andthe oxygen gas together inside the mixing chamber.

Further aspects provide an electrokinetically-altered sports beveragecomposition made according to any of the methods disclosed herein.

In particular aspects, the charge-stabilized oxygen-containingnanostructures of the electrokinetically-altered fluid comprise at leastone salt or ion from Tables 1 and 2 disclosed herein.

In certain aspects, the electrokinetically-altered sports beveragecompositions disclosed herein comprise at least one art-recognizedsports beverage ingredient. In certain aspects, they comprise a sugar orcarbohydrate, and/or caffeine.

Additionally provided are methods for enhancing physiologicalperformance and recovery time, comprising administration, to a subjectin need thereof, a electrokinetically-altered sports beveragecomposition as disclosed herein in an amount sufficient to provide forenhancing at least one of physiological performance and recovery time.In certain aspects, the methods comprise ameliorating the effects ofphysical exertion of the subject.

Additionally provided are methods for administering a sugar,carbohydrate or other sports beverage ingredient to a subject,comprising orally administering to a subject in need thereof anelectrokinetically-altered sports beverage compositions as disclosedherein, and comprising a sugar or carbohydrate, to the subject.

Further aspects provide methods for producing a sports beveragecomposition material, comprising: obtaining at least one sports beverageingredient; and combining the least one sports beverage ingredient withan electrokinetically-altered aqueous fluid comprising an ionic aqueoussolution of charge-stabilized oxygen-containing nanostructuressubstantially having an average diameter of less than about 100nanometers and stably configured in the ionic aqueous fluid in an amountsufficient to provide for enhancing at least one of physiologicalperformance and recovery time. In certain aspects, the at least onesports beverage ingredient comprises a concentrated sports beverageingredient. In certain aspects, the at least one sports beverageingredient comprises a solid sports beverage ingredient.

Additional aspects provide a method for preventing muscle damage and/orenhancing/facilitating muscle recovery from exercise (e.g., eccentricexercise), comprising administration, to a subject in need thereof, aelectrokinetically-altered sports beverage composition according to anyone of claims 1-29 in an amount sufficient to provide for preventingmuscle damage and/or enhancing/facilitating muscle recovery fromexercise. In particular aspects, the methods involve reducing biomarkersof exercise-induced muscle injury (e.g., creatine kinase (CK)). Infurther aspects, the methods comprise reducing subjective ratings ofmuscle soreness. In particular aspects, the methods comprise preservingmuscle contractile function (e.g., maximal force, joint ROM). In certainaspects, the methods comprise improving exercise performance.

Additionally provided are methods for preventing exercise-induced tendondamage and/or enhancing/facilitating tendon recovery from exerciseand/or exercise-induced damage and/or surgery, comprisingadministration, to a subject in need thereof, aelectrokinetically-altered sports beverage composition as disclosedherein in an amount sufficient to provide for preventingexercise-induced tendon damage and/or enhancing/facilitating tendonrecovery from exercise and/or exercise-induced damage and/or surgery. Incertain aspects, the methods comprise preventing or ameliorating atleast one of exercise-induced tendinosis, tendonitis, tenosynovitis, andavulsion.

Further provided are methods for preventing and/or ameliorating and/orenhancing recovery from tendon strain associated with chronic,repetitive movement, comprising administration, to a subject in needthereof, a electrokinetically-altered sports beverage as disclosedherein in an amount sufficient to provide for preventing and/orameliorating and/or enhancing recovery from tendon strain associatedwith chronic, repetitive movement.

Electrokinetically-generated Fluids:

“Electrokinetically generated fluid,” as used herein, refers toApplicants' inventive electrokinetically-generated fluids generated, forpurposes of the working Examples herein, by the exemplary Mixing Devicedescribed in detail herein (see also US 2008/0219088 (now U.S. Pat. No.7,832,920), US 2008/0281001 (now U.S. Pat. No. 7,919,534); US2010/0038244, WO 2008/052143, US 2009/0227018; WO 2009/055614, and US20100029764 (all incorporated herein by reference in their entirety fortheir teachings regarding the nature and biological activities ofelectrokinetically-altered fluids). The electrokinetic fluids, asdemonstrated by the data disclosed and presented herein, represent noveland fundamentally distinct fluids relative to prior artnon-electrokinetic fluids, including relative to prior art oxygenatednon-electrokinetic fluids (e.g., pressure pot oxygenated fluids and thelike). As disclosed in various aspects herein, theelectrokinetically-generated fluids have unique and novel physical andbiological properties including, but not limited to the following:

In particular aspects, the electrokinetically altered aqueous fluidcomprise an ionic aqueous solution of charge-stabilizedoxygen-containing nanostructures substantially having an averagediameter of less than about 100 nanometers and stably configured in theionic aqueous fluid in an amount sufficient to provide, upon contact ofa living cell by the fluid, modulation of at least one of cellularmembrane potential and cellular membrane conductivity.

In particular aspects, electrokinetically-generated fluids refers tofluids generated in the presence of hydrodynamically-induced, localized(e.g., non-uniform with respect to the overall fluid volume)electrokinetic effects (e.g., voltage/current pulses), such as devicefeature-localized effects as described herein. In particular aspectssaid hydrodynamically-induced, localized electrokinetic effects are incombination with surface-related double layer and/or streaming currenteffects as disclosed and discussed herein.

In particular aspects the administered inventiveelectrokinetically-altered fluids comprise charge-stabilizedoxygen-containing nanostructures in an amount sufficient to providemodulation of at least one of cellular membrane potential and cellularmembrane conductivity. In certain embodiments, theelectrokinetically-altered fluids are superoxygenated (e.g., RNS-20,RNS-40 and RNS-60, comprising 20 ppm, 40 ppm and 60 ppm dissolvedoxygen, respectively, in standard saline). In particular embodiments,the electrokinetically-altered fluids are not-superoxygenated (e.g.,RNS-10 or Solas, comprising 10 ppm (e.g., approx. ambient levels ofdissolved oxygen in standard saline). In certain aspects, the salinity,sterility, pH, etc., of the inventive electrokinetically-altered fluidsis established at the time of electrokinetic production of the fluid,and the sterile fluids are administered by an appropriate route.Alternatively, at least one of the salinity, sterility, pH, etc., of thefluids is appropriately adjusted (e.g., using sterile saline orappropriate diluents) to be physiologically compatible with the route ofadministration prior to administration of the fluid. Preferably, anddiluents and/or saline solutions and/or buffer compositions used toadjust at least one of the salinity, sterility, pH, etc., of the fluidsare also electrokinetic fluids, or are otherwise compatible.

In particular aspects, the inventive electrokinetically-altered fluidscomprise saline (e.g., one or more dissolved salt(s); e.g., alkali metalbased salts (Li+, Na+, K+, Rb+, Cs+, etc.), alkaline earth based salts(e.g., Mg++, Ca++), etc., or transition metal-based positive ions (e.g.,Cr, Fe, Co, Ni, Cu, Zn, etc.,), in each case along with any suitableanion components, including, but not limited to F—, Cl—, Br—, I—, PO4-,SO4-, and nitrogen-based anions. Particular aspects comprise mixed saltbased electrokinetic fluids (e.g., Na+, K+, Ca++, Mg++, transition metalion(s), etc.) in various combinations and concentrations, and optionallywith mixtures of counterions. In particular aspects, the inventiveelectrokinetically-altered fluids comprise standard saline (e.g.,approx. 0.9% NaCl, or about 0.15 M NaCl). In particular aspects, theinventive electrokinetically-altered fluids comprise saline at aconcentration of at least 0.0002 M, at least 0.0003 M, at least 0.001 M,at least 0.005 M, at least 0.01 M, at least 0.015 M, at least 0.1 M, atleast 0.15 M, or at least 0.2 M. In particular aspects, the conductivityof the inventive electrokinetically-altered fluids is at least 10 μS/cm,at least 40 μS/cm, at least 80 μS/cm, at least 100 μS/cm, at least 150μS/cm, at least 200 μS/cm, at least 300 μS/cm, or at least 500 μS/cm, atleast 1 mS/cm, at least 5, mS/cm, 10 mS/cm, at least 40 mS/cm, at least80 mS/cm, at least 100 mS/cm, at least 150 mS/cm, at least 200 mS/cm, atleast 300 mS/cm, or at least 500 mS/cm. In particular aspects, any saltmay be used in preparing the inventive electrokinetically-alteredfluids, provided that they allow for formation of biologically activesalt-stabilized nanostructures (e.g., salt-stabilized oxygen-containingnanostructures) as disclosed herein.

According to particular aspects, the biological effects of the inventivefluid compositions comprising charge-stabilized gas-containingnanostructures can be modulated (e.g., increased, decreased, tuned,etc.) by altering the ionic components of the fluids, and/or by alteringthe gas component of the fluid.

According to particular aspects, the biological effects of the inventivefluid compositions comprising charge-stabilized gas-containingnanostructures can be modulated (e.g., increased, decreased, tuned,etc.) by altering the gas component of the fluid. In preferred aspects,oxygen is used in preparing the inventive electrokinetic fluids. Inadditional aspects mixtures of oxygen along with at least one other gasselected from Nitrogen, Oxygen, Argon, Carbon dioxide, Neon, Helium,krypton, hydrogen and Xenon. As described above, the ions may also bevaried, including along with varying the gas constituent(s).

Given the teachings and assay systems disclosed herein (e.g., cell-basedcytokine assays, VO₂ max assays, RPE (rating of perceived exertion)assays, lactate assays, etc.) one of skill in the art will readily beable to select appropriate salts and concentrations thereof to achievethe biological activities disclosed herein.

TABLE 1 Exemplary cations and anions. Exemplary Cations: Name FormulaOther name(s) Aluminum Al⁺³ Ammonium NH₄ ⁺ Barium Ba⁺² Calcium Ca⁺²Chromium(II) Cr⁺² Chromous Chromium(III) Cr⁺³ Chromic Copper(I) Cu⁺Cuprous Copper(II) Cu⁺² Cupric Iron(II) Fe⁺² Ferrous Iron(III) Fe⁺³Ferric Hydrogen H⁺ Hydronium H₃O⁺ Lead(II) Pb⁺² Lithium Li⁺ MagnesiumMg⁺² Manganese(II) Mn⁺² Manganous Manganese(III) Mn⁺³ ManganicMercury(I) Hg₂ ⁺² Mercurous Mercury(II) Hg⁺² Mercuric Nitronium NO₂ ⁺Potassium K⁺ Silver Ag⁺ Sodium Na⁺ Strontium Sr⁺² Tin(II) Sn⁺² StannousTin(IV) Sn⁺⁴ Stannic Zinc Zn⁺² Exemplary Anions: Simple ions: Hydride H⁻Fluoride F⁻ Chloride Cl⁻ Bromide Br⁻ Iodide I⁻ Oxide O²⁻ Sulfide S²⁻Nitride N³⁻ Oxoanions: Arsenate AsO₄ ³⁻ Arsenite AsO₃ ³⁻ Sulfate SO₄ ²⁻Hydrogen sulfate HSO₄ ⁻ Thiosulfate S₂O₃ ²⁻ Sulfite SO₃ ²⁻ PerchlorateClO₄ ⁻ Chlorate ClO₃ ⁻ Chlorite ClO₂ ⁻ Hypochlorite OCl⁻ Carbonate CO₃²⁻ Hydrogen carbonate HCO₃ ⁻ or Bicarbonate Phosphate PO₄ ³⁻ Hydrogenphosphate HPO₄ ²⁻ Dihydrogen phosphate H₂PO₄ ⁻ Nitrate NO₃ ⁻ Nitrite NO₂⁻ Iodate IO₃ ⁻ Bromate BrO₃ ⁻ Hypobromite OBr⁻ Chromate CrO₄ ²⁻Dichromate Cr₂O₇ ²⁻ Anions from Organic Acids: Acetate CH₃COO⁻ formateHCOO⁻ Others: Cyanide CN⁻ Cyanate OCN⁻ Thiocyanate SCN⁻ Hydroxide OH⁻Amide NH₂ ⁻ Peroxide O₂ ²⁻ Oxalate C₂O₄ ²⁻ Permanganate MnO₄ ⁻

TABLE 2 Exemplary cations and anions. Formula Charge Name MonoatomicCations H⁺ 1+ hydrogen ion Li⁺ 1+ lithium ion Na⁺ 1+ sodium ion K⁺ 1+potassium ion Cs⁺ 1+ cesium ion Ag⁺ 1+ silver ion Mg²⁺ 2+ magnesium ionCa²⁺ 2+ calcium ion Sr²⁺ 2+ strontium ion Ba²⁺ 2+ barium ion Zn²⁺ 2+zinc ion Cd²⁺ 2+ cadmium ion Al³⁺ 3+ aluminum ion Polyatomic Cations NH₄⁺ 1+ ammonium ion H₃O⁺ 1+ hydronium ion Multivalent Cations Cr²⁺ 2chromium(II) or chromous ion Cr³⁺ 3 chromium(III)or chromic ion Mn²⁺ 2manganese(II) or manganous ion Mn⁴⁺ 4 manganese(IV) ion Fe²⁺ 2 iron(II)or ferrous ion Fe³⁺ 3 iron(III) or ferric ion Co²⁺ 2 cobalt(II) orcobaltous ion Co³⁺ 3 cobalt(II) or cobaltic ion Ni²⁺ 2 nickel(II) ornickelous ion Ni³⁺ 3 nickel(III) or nickelic ion Cu⁺ 1 copper(I) orcuprous ion Cu²⁺ 2 copper(II) or cupric ion Sn²⁺ 2 tin(II) or atannousion Sn⁴⁺ 4 tin(IV) or atannic ion Pb²⁺ 2 lead(II) or plumbous ion Pb⁴⁺ 4lead(IV) or plumbic ion Monoatomic Anions H⁻ 1− hydride ion F⁻ 1−fluoride ion Cl⁻ 1− chloride ion Br⁻ 1− bromide ion I⁻ 1− iodide ion O²⁻2− oxide ion S²⁻ 2− sulfide ion N³⁻ 3− nitride ion Polyatomic Anions OH⁻1− hydroxide ion CN⁻ 1− cyanide ion SCN⁻ 1− thiocyanate ion C₂H₃O₂ ⁻ 1−acetate ion ClO⁻ 1− hypochlorite ion ClO₂ ⁻ 1− chlorite ion ClO₃ ⁻ 1−chlorate ion ClO₄ ⁻ 1− perchlorate ion NO₂ ⁻ 1− nitrite ion NO₃ ⁻ 1−nitrate ion MnO₄ ²⁻ 2− permanganate ion CO₃ ²⁻ 2− carbonate ion C₂O₄ ²⁻2− oxalate ion CrO₄ ²⁻ 2− chromate ion Cr₂O₇ ²⁻ 2− dichromate ion SO₃ ²⁻2− sulfite ion SO₄ ²⁻ 2− sulfate ion PO₃ ³⁻ 3− phosphite ion PO₄ ³⁻ 3−phosphate ion

The present disclosure sets forth novel electrokinetically-generatedgas-enriched fluids, including, but not limited toelectrokinetically-generated gas-enriched water, ionic water, aqueoussolutions, beverages, sports drinks, energy drinks, food drinks, aqueoussaline solutions (e.g., standard aqueous saline solutions, and othersaline solutions as discussed herein and as would be recognized in theart, including any physiological compatible saline solutions).

In particular aspects, the electrokinetically altered aqueous fluids aresuitable to modulate ¹³C-NMR line-widths of reporter solutes (e.g.,trehelose) dissolved therein.

NMR line-width effects are in indirect method of measuring, for example,solute ‘tumbling’ in a test fluid as described herein in particularworking Examples.

In particular aspects, the electrokinetically altered aqueous fluids arecharacterized by at least one of: distinctive square wave voltametrypeak differences at any one of −0.14V, −0.47V, −1.02V and −1.36V;polarographic peaks at −0.9 volts; and an absence of polarographic peaksat −0.19 and −0.3 volts, which are unique to the electrokineticallygenerated fluids as disclosed herein in particular working Examples.

In particular aspects, the electrokinetically altered aqueous fluids aresuitable to alter at least one of cellular membrane potential andcellular membrane conductivity (e.g., a voltage-dependent contributionof the whole-cell conductance as measure in patch clamp studiesdisclosed herein).

In particular aspects, the electrokinetically altered aqueous fluids aregasified (e.g., oxygenated), wherein the gas (e.g., oxygen) in the fluidis present in an amount of at least 15, ppm, at least 25 ppm, at least30 ppm, at least 40 ppm, at least 50 ppm, or at least 60 ppm dissolvedgas (e.g., oxygen) at atmospheric pressure. In particular aspects, theelectrokinetically altered aqueous fluids have less than 15 ppm, lessthat 10 ppm of dissolved gas (e.g., oxygen) at atmospheric pressure, orapproximately ambient oxygen levels.

In particular aspects, the electrokinetically altered aqueous fluids areoxygenated, wherein the gas (e.g., oxygen) in the fluid is present in anamount between approximately 8 ppm and approximately 15 ppm, and in thiscase is sometimes referred to herein as “Solas” or Solas-based fluids.

In particular aspects, the electrokinetically altered aqueous fluidcomprises at least one of solvated electrons (e.g., stabilized bymolecular oxygen), and electrokinetically modified and/or charged gas(e.g., oxygen) species, and wherein in certain embodiments the solvatedelectrons and/or electrokinetically modified or charged gas (e.g.,oxygen)(species are present in an amount of at least 0.01 ppm, at least0.1 ppm, at least 0.5 ppm, at least 1 ppm, at least 3 ppm, at least 5ppm, at least 7 ppm, at least 10 ppm, at least 15 ppm, or at least 20ppm.

In particular aspects, the electrokinetically altered aqueous fluids aresuitable to alter cellular membrane structure or function (e.g.,altering of a conformation, ligand binding activity, or a catalyticactivity of a membrane associated protein) sufficient to provide formodulation of intracellular signal transduction, wherein in particularaspects, the membrane associated protein comprises at least one selectedfrom the group consisting of receptors, transmembrane receptors (e.g.,G-Protein Coupled Receptor (GPCR), TSLP receptor, beta 2 adrenergicreceptor, bradykinin receptor, etc.), ion channel proteins,intracellular attachment proteins, cellular adhesion proteins, andintegrins. In certain aspects, the effected G-Protein Coupled Receptor(GPCR) interacts with a G protein α subunit (e.g., Gα_(s), Gα_(i),Gα_(q), and Gα₁₂).

In particular aspects, the electrokinetically altered aqueous fluids aresuitable to modulate intracellular signal transduction, comprisingmodulation of a calcium dependant cellular messaging pathway or system(e.g., modulation of phospholipase C activity, or modulation ofadenylate cyclase (AC) activity).

In particular aspects, the electrokinetically altered aqueous fluids arecharacterized by various biological activities (e.g., regulation ofcytokines, receptors, enzymes and other proteins and intracellularsignaling pathways) described in the working Examples and elsewhereherein.

In particular aspects, the electrokinetically altered aqueous fluidsreduce DEP-induced TSLP receptor expression in bronchial epithelialcells (BEC).

In particular aspects, the electrokinetically altered aqueous fluidsinhibit the DEP-induced cell surface-bound MMP9 levels in bronchialepithelial cells (BEC).

In particular aspects, the physical and biological effects (e.g., theability to alter cellular membrane structure or function sufficient toprovide for modulation of intracellular signal transduction) of theelectrokinetically altered aqueous fluids persists for at least two, atleast three, at least four, at least five, at least 6 months, or longerperiods, in a closed container (e.g., closed gas-tight container),preferably at 4° C.

Therefore, further aspects provide said electrokinetically-generatedsolutions and methods of producing an electrokinetically alteredgasified (e.g., oxygenated) aqueous fluid or solution, comprising:providing a flow of a fluid material between two spaced surfaces inrelative motion and defining a mixing volume therebetween, wherein thedwell time of a single pass of the flowing fluid material within andthrough the mixing volume is greater than 0.06 seconds or greater than0.1 seconds; and introducing oxygen (O₂) into the flowing fluid materialwithin the mixing volume under conditions suitable to dissolve at least20 ppm, at least 25 ppm, at least 30, at least 40, at least 50, or atleast 60 ppm gas (e.g., oxygen) into the material, andelectrokinetically alter the fluid or solution. In certain aspects, thegas (e.g., oxygen) is infused into the material in less than 100milliseconds, less than 200 milliseconds, less than 300 milliseconds, orless than 400 milliseconds. In particular embodiments, the ratio ofsurface area to the volume is at least 12, at least 20, at least 30, atleast 40, or at least 50.

Yet further aspects, provide a method of producing an electrokineticallyaltered gasified (e.g., oxygenated) aqueous fluid or solution,comprising: providing a flow of a fluid material between two spacedsurfaces defining a mixing volume therebetween; and introducing gas(e.g., oxygen) into the flowing material within the mixing volume underconditions suitable to infuse at least 20 ppm, at least 25 ppm, at least30, at least 40, at least 50, or at least 60 ppm gas (e.g., oxygen) intothe material in less than 100 milliseconds, less than 200 milliseconds,less than 300 milliseconds, or less than 400 milliseconds. In certainaspects, the dwell time of the flowing material within the mixing volumeis greater than 0.06 seconds or greater than 0.1 seconds. In particularembodiments, the ratio of surface area to the volume is at least 12, atleast 20, at least 30, at least 40, or at least 50.

Additional embodiments provide a method of producing anelectrokinetically altered gasified (e.g., oxygenated) aqueous fluid orsolution, comprising use of a mixing device for creating an outputmixture by mixing a first material and a second material, the devicecomprising: a first chamber configured to receive the first materialfrom a source of the first material; a stator; a rotor having an axis ofrotation, the rotor being disposed inside the stator and configured torotate about the axis of rotation therein, at least one of the rotor andstator having a plurality of through-holes; a mixing chamber definedbetween the rotor and the stator, the mixing chamber being in fluidcommunication with the first chamber and configured to receive the firstmaterial therefrom, and the second material being provided to the mixingchamber via the plurality of through-holes formed in the one of therotor and stator; a second chamber in fluid communication with themixing chamber and configured to receive the output material therefrom;and a first internal pump housed inside the first chamber, the firstinternal pump being configured to pump the first material from the firstchamber into the mixing chamber. In certain aspects, the first internalpump is configured to impart a circumferential velocity into the firstmaterial before it enters the mixing chamber. Further embodimentsprovide a method of producing an electrokinetically altered gasified(e.g., oxygenated) aqueous fluid or solution, comprising use of a mixingdevice for creating an output mixture by mixing a first material and asecond material, the device comprising: a stator; a rotor having an axisof rotation, the rotor being disposed inside the stator and configuredto rotate about the axis of rotation therein; a mixing chamber definedbetween the rotor and the stator, the mixing chamber having an openfirst end through which the first material enters the mixing chamber andan open second end through which the output material exits the mixingchamber, the second material entering the mixing chamber through atleast one of the rotor and the stator; a first chamber in communicationwith at least a majority portion of the open first end of the mixingchamber; and a second chamber in communication with the open second endof the mixing chamber.

Additional aspects provide an electrokinetically altered gasified (e.g.,oxygenated) aqueous fluid or solution made according to any of the abovemethods.

The phrases “enhancing exercise performance” and “enhancing recoverytime” refer to, and include but limited to, reversing, alleviating,inhibiting the progress of, or preventing a negative or limitingcondition or symptom of exercise (e.g., exercise stress and othervariable as discussed and disclosed herein), and/or enhancing a positiveor beneficial condition or symptom of exercise and/or enhancing and/orspeeding recovery from such negative or limiting conditions or symptomsof exercise (e.g., exercise stress), as various taught and/or definedherein.

The phrases “performance enhancing amount” and “recovery enhancingamount” refer to, and include, administration (or consumption) an amountof the electrokinetically-altered fluids and beverages disclosed hereinthat is sufficient to reverse, alleviate, inhibit the progress of, orprevent a negative or limiting condition or symptom of exercise (e.g.,exercise stress), and/or enhance a positive or beneficial condition orsymptom of exercise, and/or enhanced and/or accelerated recovery fromsuch negative or limiting conditions or symptoms of exercise (e.g.,exercise stress), as various taught and/or defined herein.

Electrokinetically Gas (e.q., Oxygen)-Enriched Aqueous Fluids andSolutions

In the Figures and Examples that follow, it is to be understood thatwhile oxygen is the preferred gas used in producing the inventiveelectrokinetic fluids, and the describe biological activities, thepresent invention encompasses other electrokinetically-altered fluidscomprising the use of various positive ions and counter ions asdiscussed herein above, and including other additives as will befamiliar to those of ordinary skill in the beverage art (e.g., sportsbeverage, energy beverage, etc.). In certain aspects, suchelectrokinetic fluid variants have the ability to modulate at least oneof cellular membrane potential or cellular membrane conductance, and/ormodulate at least one biological activity as taught herein with theexemplary oxygen embodiments that comprise charge-stabilizedoxygen-containing nanostructures substantially having an averagediameter less than about 100 nm.

Charge-stabilized Nanostructures (e.g., Charge StabilizedOxygen-containing Nanostructures):

As described in detail in US 2008/0219088 (now U.S. Pat. No. 7,832,920),US 2008/0281001 (now U.S. Pat. No. 7,919,534); US 2010/0038244, WO2008/052143, US 2009/0227018; WO 2009/055614, and US 20100029764 (allincorporated herein by reference in their entirety for their teachingsregarding the nature and biological activities ofelectrokinetically-altered fluids, and see particularly under “DoubleLayer Effect,” “Dwell Time,” “Rate of Infusion,” and “Bubble sizeMeasurements,” thereof) the electrokinetic mixing device creates, in amatter of milliseconds, a unique non-linear fluid dynamic interaction ofthe first material (e.g., water, saline, etc.) and the second material(e.g., gas, such as oxygen, etc.) with complex, dynamic turbulenceproviding complex mixing in contact with an effectively enormous surfacearea (including those of the device and of the exceptionally small gasbubbles of less that 100 nm) including certain surface features thatprovide for the novel electrokinetic effects. The, feature-localizedelectrokinetic effects have been demonstrated using a specially designedmixing device comprising insulated rotor and stator features (Id).

As well-recognized in the art, charge redistributions and/or solvatedelectrons are known to be highly unstable in aqueous solution. Accordingto particular aspects, Applicants' electrokinetic effects (e.g., chargeredistributions, including, in particular aspects, solvated electrons)are surprisingly stabilized within the output material (e.g., water,saline solutions, ionic solutions, beverage solutions, etc.). In fact,the stability of the properties and biological activity of the inventiveelectrokinetic fluids can be maintained for months in a gas-tightcontainer (preferably at 4° C., indicating involvement of dissolved gas(e.g., oxygen) in helping to generate and/or maintain, and/or mediatethe properties and activities of the inventive solutions. Significantly,the charge redistributions and/or solvated electrons are stablyconfigured in the inventive electrokinetic ionic aqueous fluids in anamount sufficient to provide, upon contact with a living cell (e.g.,mammalian cell) by the fluid, modulation of at least one of cellularmembrane potential and cellular membrane conductivity (see, e.g., US2009/0227018; WO 2009/055614, and US 20100029764, all incorporated byreference herein in the entirety for their teachings on the nature andbiological activity of electrokinetically-altered fluids).

According to particular aspects, interactions between the watermolecules and the molecules of the substances (e.g., oxygen) dissolvedin the water change the collective structure of the water and providefor nanoscale structures (e.g., caged oxygen structures or clusters),including charge-stabilized nanostructures comprising oxygen and/orstabilized and/or associated charges (e.g., nanostructures comprisinggas along with ions and/or electrons) imparted to the inventive outputmaterials. Without being bound by mechanism, and according to theproperties and activities described herein, the configuration of thenanostructures in particular aspects is such that they: comprise (atleast for formation and/or stability and/or biological activity)dissolved gas (e.g., oxygen); enable the electrokinetic fluids (e.g.,hydration beverages, sports beverages, performance beverages, energybeverages, etc.) to modulate (e.g., impart or receive) charges and/orcharge effects (e.g., modulation of membrane potential and/orconductivity) upon contact or sufficient proximity with a cell membraneor related constituent thereof; and in particular aspects provide forstabilization (e.g., carrying, harboring, trapping) solvated electronsand/or electric fields in a biologically-relevant form.

According to particular aspects, in ionic or saline (e.g., water,saline, standard saline, etc.) solutions, the inventive nanostructurescomprise charge stabilized nanostructures (e.g., average diameter lessthat 100 nm) that may comprise at least one dissolved gas molecule(e.g., oxygen) within a charge-stabilized hydration shell. According toadditional aspects, and as described elsewhere herein, thecharge-stabilized hydration shell may comprise a cage or void harboringthe at least one dissolved gas molecule (e.g., oxygen). According tofurther aspects, by virtue of the provision of suitablecharge—stabilized hydration shells, the charge-stabilized nanostructureand/or charge-stabilized oxygen containing nano-structures mayadditionally comprise a solvated electron (e.g., stabilized solvatedelectron).

Without being bound by mechanism or particular theory, charge-stabilizedmicrobubbles stabilized by ions in aqueous liquid in equilibrium withambient (atmospheric) gas have been proposed (Bunkin, et al., Journal ofExperimental and Theoretical Physics, 104:486-498, 2007; incorporatedherein by reference in its entirety). According to particular aspects ofthe present invention, Applicants' novel electrokinetic fluids comprisea novel, biologically active form of charge-stabilized oxygen-containingnanostructures, and may further comprise novel arrays, clusters orassociations of such structures.

According to the charge-stabilized microbubble model, the short-rangemolecular order of the water structure is destroyed by the presence of agas molecule (e.g., a dissolved gas molecule initially complexed with anonadsorptive ion provides a short-range order defect), providing forcondensation of ionic droplets, wherein the defect is surrounded byfirst and second coordination spheres of water molecules, which arealternately filled by adsorptive ions (e.g., acquisition of a ‘screeningshell of Na⁺ ions to form an electrical double layer) and nonadsorptiveions (e.g., Cl⁻ ions occupying the second coordination sphere) occupyingsix and 12 vacancies, respectively, in the coordination spheres. Inunder-saturated ionic solutions (e.g., undersaturated saline solutions),this hydrated ‘nucleus’ remains stable until the first and secondspheres are filled by six adsorptive and five nonadsorptive ions,respectively, and then undergoes Coulomb explosion creating an internalvoid containing the gas molecule, wherein the adsorptive ions (e.g., Na⁺ions) are adsorbed to the surface of the resulting void, while thenonadsorptive ions (or some portion thereof) diffuse into the solution(Bunkin et al., supra). In this model, the void in the nanostructure isprevented from collapsing by Coulombic repulsion between the ions (e.g.,Na⁺ ions) adsorbed to its surface. The stability of the void-containingnanostructures is postulated to be due to the selective adsorption ofdissolved ions with like charges onto the void/bubble surface anddiffusive equilibrium between the dissolved gas and the gas inside thebubble, where the negative (outward electrostatic pressure exerted bythe resulting electrical double layer provides stable compensation forsurface tension, and the gas pressure inside the bubble is balanced bythe ambient pressure. According to the model, formation of suchmicrobubbles requires an ionic component, and in certain aspectscollision-mediated associations between particles may provide forformation of larger order clusters (arrays) (Id).

The charge-stabilized microbubble model of Bunkin et al., suggests thatthe particles can be gas microbubbles, but contemplates only spontaneousformation of such structures in ionic solution in equilibrium withambient air, is uncharacterized and silent as to whether oxygen iscapable of forming such structures, how such structures might be furtherstabilized, and is likewise silent as to whether solvated electronsmight be associated and/or stabilized by such structures.

According to particular aspects of the present invention, the inventiveelectrokinetic fluids comprising charge-stabilized nanostructures and/orcharge-stabilized oxygen-containing nanostructures are novel andfundamentally distinct from the postulated non-electrokinetic,atmospheric charge-stabilized microbubble structures according to themicrobubble model. Significantly, this conclusion is unavoidable,deriving, at least in part, from the fact that control saline solutionsdo not have the biological properties disclosed herein, whereasApplicants' charge-stabilized nanostructures provide a novel,biologically active form of charge-stabilized oxygen-containingnanostructures.

According to particular aspects of the present invention, Applicants'novel electrokinetic device and methods provide for novelelectrokinetically-altered fluids comprising significant quantities ofcharge-stabilized nanostructures in excess of any amount that may or maynot spontaneously occur in ionic fluids in equilibrium with air, or inany non-electrokinetically generated fluids. In particular aspects, thecharge-stabilized nanostructures comprise charge-stabilizedoxygen-containing nanostructures. In additional aspects, thecharge-stabilized nanostructures are all, or substantially allcharge-stabilized oxygen-containing nanostructures, or thecharge-stabilized oxygen-containing nanostructures is the majorcharge-stabilized gas-containing nanostructure species in theelectrokinetic fluid.

According to yet further aspects, the charge-stabilized nanostructuresand/or the charge-stabilized oxygen-containing nanostructures maycomprise or harbor a solvated electron and/or electric field (electricdouble layer), and thereby provide a novel stabilized solvated electron,or electric field carrier. In particular aspects, the charge-stabilizednanostructures and/or the charge-stabilized oxygen-containingnanostructures provide a novel type of electride (or invertedelectride), which in contrast to conventional solute electrides having asingle organically coordinated cation, rather have a plurality ofcations stably arrayed about a void or a void containing an oxygen atom,wherein the arrayed sodium ions are coordinated by water hydrationshells, rather than by organic molecules. According to particularaspects, a solvated electron and or electric field (electric doublelayer) may be accommodated by the hydration shell of water molecules, ora hydrated electron may be accommodated within the nanostructure voidand distributed over all the cations. In certain aspects, therefore, theinventive nanostructures provide a novel ‘super electride’ structure insolution by not only providing for distribution/stabilization of thesolvated electron and/or electric field over multiple arrayed sodiumcations, but also providing for association or partial association of asolvated electron with the caged oxygen molecule(s) in the void—thesolvated electron distributing over an array of sodium atoms and atleast one oxygen atom. According to particular aspects, therefore,‘solvated electrons’ in the inventive electrokinetic fluids, may not besolvated in the traditional model comprising direct hydration by watermolecules. Alternatively, in limited analogy with dried electride salts,solvated electrons in the inventive electrokinetic fluids may bedistributed over multiple charge-stabilized nanostructures to provide a‘lattice glue’ to stabilize higher order arrays in aqueous solution.

In particular aspects, the inventive charge-stabilized nanostructuresand/or the charge-stabilized oxygen-containing nanostructures arecapable of interacting with cellular membranes or constituents thereof,or proteins, etc., to mediate biological activities. In particularaspects, the inventive charge-stabilized nanostructures and/or thecharge-stabilized oxygen-containing nanostructures harboring a solvatedelectron and/or electric field (electric double layer) are capable ofinteracting with cellular membranes or constituents thereof, orproteins, etc., to mediate biological activities.

In particular aspects, the inventive charge-stabilized nanostructuresand/or the charge-stabilized oxygen-containing nanostructures interactwith cellular membranes or constituents thereof, or proteins, etc., as acharge and/or charge effect donor (delivery) and/or as a charge and/orcharge effect recipient to mediate biological activities. In particularaspects, the inventive charge-stabilized nanostructures and/or thecharge-stabilized oxygen-containing nanostructures harboring a solvatedelectron and/or electric field (electric double layer) interact withcellular membranes as a charge and/or charge effect donor and/or as acharge and/or charge effect recipient to mediate biological activities.

In particular aspects, the inventive charge-stabilized nanostructuresand/or the charge-stabilized oxygen-containing nanostructures areconsistent with, and account for the observed stability and biologicalproperties of the inventive electrokinetic fluids, and further provide anovel electride (or inverted electride) that provides for stabilizedsolvated electrons and/or electric fields (e.g., electric double layers)in aqueous ionic solutions (e.g., water, saline solutions, beverages,etc.).

In particular aspects, the charge-stabilized oxygen-containingnanostructures substantially comprise, take the form of, or can giverise to, charge-stabilized oxygen-containing nanobubbles. In particularaspects, charge-stabilized oxygen-containing clusters provide forformation of relatively larger arrays of charge-stabilizedoxygen-containing nanostructures, and/or charge-stabilizedoxygen-containing nanobubbles or arrays thereof. In particular aspects,the charge-stabilized oxygen-containing nanostructures can provide forformation of hydrophobic nanobubbles upon contact with a hydrophobicsurface.

In particular aspects, the charge-stabilized oxygen-containingnanostructures substantially comprise at least one oxygen molecule. Incertain aspects, the charge-stabilized oxygen-containing nanostructuressubstantially comprise at least 1, at least 2, at least 3, at least 4,at least 5, at least 10 at least 15, at least 20, at least 50, at least100, or greater oxygen molecules. In particular aspects,charge-stabilized oxygen-containing nanostructures comprise or give riseto surface nanobubbles (e.g., hydrophobic nanobubbles) of about 20nm×1.5 nm, comprise about 12 oxygen molecules (e.g., based on the sizeof an oxygen molecule (approx 0.3 nm by 0.4 nm), assumption of an idealgas and application of n=PV/RT, where P=1 atm, R=0.0820571.atm/mol.K;T=295K; V=pr²h=4.7×10⁻²² L , where r=10×10⁻⁹ m, h=1.5×10⁻⁹ m, andn=1.95×10⁻²² moles).

In certain aspects, the percentage of oxygen molecules present in thefluid that are in charge-stabilized nanostructures, or arrays thereof,having a charge-stabilized configuration in the ionic aqueous fluid is apercentage amount selected from the group consisting of greater than:0.1%, 1%; 2%; 5%; 10%; 15%; 20%; 25%; 30%; 35%; 40%; 45%; 50%; 55%; 60%;65%; 70%; 75%; 80%; 85%; 90%; and greater than 95%. Preferably, thispercentage is greater than about 5%, greater than about 10%, greaterthan about 15%f, or greater than about 20%. In additional aspects, thesubstantial, or biologically relevant size (average or mean diameter) ofthe charge-stabilized oxygen-containing nanostructures, or arraysthereof, having a charge-stabilized configuration in the ionic aqueousfluid is a size selected from the group consisting of less than: 100 nm;90 nm; 80 nm; 70 nm; 60 nm; 50 nm; 40 nm; 30 nm; 20 nm; 10 nm; 5 nm; 4nm; 3 nm; 2 nm; and 1 nm. Preferably, this size is less than about 50nm, less than about 40 nm, less than about 30 nm, less than about 20 nm,or less than about 10 nm.

In certain aspects, the inventive electrokinetic fluids comprisesolvated electrons and/or stabilized electric fields. In furtheraspects, the inventive electrokinetic fluids comprises charge-stabilizednanostructures and/or charge-stabilized oxygen-containingnanostructures, and/or arrays thereof, which comprise at least one of:solvated electron(s); and unique charge distributions (polar, symmetric,asymmetric charge distribution) or electric field. In certain aspects,the charge-stabilized nanostructures and/or charge-stabilizedoxygen-containing nanostructures, and/or arrays thereof, haveparamagnetic properties.

By contrast, relative to the inventive electrokinetic fluids, controlpressure pot oxygenated fluids (non-electrokinetic fluids) and the likedo not comprise such charge-stabilized biologically-activenanostructures and/or biologically-active charge-stabilizedoxygen-containing nanostructures and/or arrays thereof, capable ofmodulation of at least one of cellular membrane potential and cellularmembrane conductivity.

In additional aspects, the inventive electrokinetic fluids comprisecharge-stabilized nanostructures as disclosed herein, comprisingvariations in at least one of the ionic components (e.g., variations inthe cation(s) or counterion(s)) and the gas component(s). As describedelsewhere herein, in particular aspects, the inventiveelectrokinetically-altered fluids comprise saline (e.g., one or moredissolved salt(s); e.g., alkali metal based salts (Li+, Na+, K+, Rb+,Cs-F, etc.), alkaline earth based salts (e.g., Mg++, Ca++), etc., ortransition metal-based positive ions (e.g., Cr, Fe, Co, Ni, Cu, Zn,etc.,), in each case along with any suitable anion components,including, but not limited to F—, Cl—, Br—, I—, PO4-, SO4-, andnitrogen-based anions. . Particular aspects comprise mixed salt basedelectrokinetic fluids (e.g., Na+, K+, Ca++, Mg++, transition metalion(s), etc.) in various combinations and concentrations, and optionallywith mixtures of counterions. In particular aspects, the inventiveelectrokinetically-altered fluids comprise standard saline (e.g.,approx. 0.9% NaCl, or about 0.15 M NaCl). In particular aspects, theinventive electrokinetically-altered fluids comprise saline at aconcentration of at least 0.0002 M, at least 0.0003 M, at least 0.001 M,at least 0.005 M, at least 0.01 M, at least 0.015 M, at least 0.1 M, atleast 0.15 M, or at least 0.2 M. In particular aspects, the conductivityof the inventive electrokinetically-altered fluids is at least 10 μS/cm,at least 40 μS/cm, at least 80 μS/cm, at least 100 μS/cm, at least 150μS/cm, at least 200 μS/cm, at least 300 μS/cm, or at least 500 μS/cm, atleast 1 mS/cm, at least 5 mS/cm, 10 mS/cm, at least 40 mS/cm, at least80 mS/cm, at least 100 mS/cm, at least 150 mS/cm, at least 200 mS/cm, atleast 300 mS/cm, or at least 500 mS/cm. In particular aspects, any saltmay be used in preparing the inventive electrokinetically-alteredfluids, provided that they allow for formation of biologically activesalt-stabilized nanostructures (e.g., salt-stabilized oxygen-containingnanostructures) as disclosed herein.

According to particular aspects, the biological effects of the inventivefluid compositions comprising charge-stabilized gas-containingnanostructures can be modulated (e.g., increased, decreased, tuned,etc.) by altering the ionic components of the fluids, and/or by alteringthe gas component of the fluid.

According to particular aspects, the biological effects of the inventivefluid compositions comprising charge-stabilized gas-containingnanostructures can be modulated (e.g., increased, decreased, tuned,etc.) by altering the gas component of the fluid. In preferred aspects,oxygen is used in preparing the inventive electrokinetic fluids. Inadditional aspects mixtures of oxygen along with at least one other gasselected from Nitrogen, Oxygen, Argon, Carbon dioxide, Neon, Helium,krypton, hydrogen and Xenon. As described above, the ionic components(cation(s) and and/or counterion(s)) may also be varied, including alongwith varying the gas constituent(s).

According to particular aspects, the inventiveelectrokinetically-altered fluid compositions can be formulated asorally consumable beverages/drinks (e.g., water, sports drinks, exercisedrinks, energy drinks, hydration beverages, food beverages, etc.).According to particular aspects, such beverages may have one or moreadditives, such as those already widely recognized in the relevant arts.

For example, a food beverage or fluid related to the present inventionis a food in which a liquid or a food comprising the inventiveelectrokinetically-altered fluid compositions. Since foods can be mixedwith a liquid or a food additive under various categories, such as anagricultural food, a livestock food, a fishery food, a fermented food, acanned food, an instant food and the like, according to states and formsof respective food additives, and no specific limitation is imposed on akind, and state and form of food related to the present invention. Stillfurther examples of food beverages of the present invention may includenutritional supplements and the like such as health foods in states andforms including liquid, powder, a tablet, a capsule, in which theinventive liquid or food additive is incorporated.

Beverages related to the present invention are, for example, beveragesin which an electrokinetically-altered fluid of the present invention isadded as a feature. Electrokinetically-altered fluid can be added to, orformulated as various kinds of beverages according to a kind, state andform thereof, no specific limitation is imposed on a kind, state andform of beverage. Examples thereof that can be named include alcoholicbeverages, soft beverages or refreshing beverages such as fruit juice,concentrated fruit juice, nectar, soda pop, cola beverage, teas, coffee,black tea, water, sports drinks, exercise drinks, energy drinks,hydration beverages, food beverages, and the like. Alternatively,preformed beverages can be electrokinetically processed to produce thedescribed biologically-active beverages.

According to certain aspects, the electrokinetically-altered fluids areproduced as a sports, energy and/or food drink. According to furtheraspects, the sports, energy and/or food drink made withelectrokinetically-altered fluids can contain additional components.These additional components can be added for many desirable traits,including but not limited to, mouth feel, taste, and increasednutrients. For example, additional components can be juice,carbohydrates, (e.g., mono- di- and polysaccharides, including but notlimited to sucrose, glucose, fructose, dextrose, mannose, galactose,maltose lactose, maltodextrins, glucose polymers, maltotriose, highfructose corn syrup, beet sugar, cane sugar, and sucanat ketohexosessuch sugars being arabinose, ribose, fructose, sorbose, tagatosesorbitol and those described in European Patent SpecificationPublication No. 223,540 incorporated herein by reference) salts,(including but not limited to those made from sodium, potassium,chloride, phosphorus, magnesium, calcium, sodium chloride, potassiumphosphate, potassium citrate, magnesium succinate, calcium pantothenate,sodium acetate, acidic sodium citrate, acidic sodium phosphate, sodiumbicarbonate, sodium bromide, sodium citrate, sodium lactate, sodiumphosphate, anhydrous sodium sulphate, sodium sulphate, sodium tartrate,sodium benzoate, sodium selenite, potassium chloride, potassium acetate,potassium bicarbonate, potassium bromide, potassium citrate,potassium-D-gluconate, monobasic potassium phosphate, potassiumtartrate, potassium sorbate, potassium iodide, magnesium chloride,magnesium oxide, magnesium sulphate, magnesium carbonate, magnesiumaspartate and magnesium silicate) (European Patent ApplicationPublication No. 587,972 , incorporated herein by reference, provides anextensive discussion of such salts and suitable concentrations thereof)vitamins, (e.g., vitamin C, the B vitamins, vitamin E, pantothenic acid,thiamin, niacin, niacinamide, riboflavin, iron and biotin) minerals,(e.g., chromium, magnesium and zinc) amino acids, (e.g. alanine,glycine, tryptophan, cysteine, taurine, tyrosine, histidine andarginine) electrolytes, trace elements, flavoring aids, phosphoric acid,citric acid, malic acid, fumaric acid, adipic acid, gluconic acid,lactic acid, calcium or sodium caseinate, whey protein, whey proteinconcentrate, whey protein isolate, whey protein hydrolyzate,demineralized whey protein, milk protein, soy protein, soy proteinisolate, soy protein concentrate, pea protein, rice protein, caseinhydrolyzate, soy flour, rice protein, wheat protein, corn protein andyeast concentrate.

Other ingredients including, but not limited to, coloring, flavor,artificial sweeteners and preservatives may also be added. Suitableamounts and types of all ingredients described herein are known in, theart and are not described in detail herein. It is within the skill ofone in the art to prepare a beverage formulation having suitableconcentrations of all the components.

Other component constituents of the nutritional composition in dry andliquid form include flavor components and/or colorant components. Theflavor component for the nutritional composition of the presentinvention is provided to impart a particular and characteristic tasteand sometimes an aroma to the nutritional composition. The use of aflavor component in the nutritional composition also provides anenhanced aesthetic quality to the nutritional composition which willincrease the user's appeal in using the product, including fruitflavoring. The flavor component is selected from the group consisting ofwater soluble natural or artificial extracts that include apple, banana,cherry, cinnamon, cranberry, grape, honeydew, honey, kiwi, lemon, lime,orange, peach, peppermint, pineapple, raspberry, tangerine, watermelon,wild cherry, and equivalents and combinations thereof.

The colorant component for the nutritional composition of the presentinvention is provided to impart a characteristic color in conjunctionwith a particular flavor to the nutritional composition. For example, ayellow color is used for a banana flavor, or a red color for a cherryflavor. The colorant component is selected from the group consisting ofwater soluble natural or artificial dyes that include FD&C dyes (food,drug and cosmetic use dyes) of blue, green, orange, red, yellow andviolet; iron oxide dyes; ultramarine pigments of blue, pink, red andviolet; and equivalents thereof. The dyes discussed above are wellknown, and are commercially available materials, with their chemicalstructure being described, e.g., in 21 C.F.R. Part 74 (as revised Apr.1, 1988) and the CTFA Cosmetic Ingredient Handbook, (1988), published bythe Cosmetics, Toiletry and Fragrancy Association, Inc.

Different compositions for beverages have been described in relatedreferences. U.S. Pat. No. 3,657,424, issued to Donald, et al., on Apr.18, 1972, teaches a citrus juice fortified with sodium, calcium andchloride ions beyond what are naturally present in the juice. The ionsare added to supplement the requirements of individuals havingdiminished amounts of these substances present in his or her bodyfluids. This patent is incorporated herein by reference in its entirety,including for its teaching regarding the addition of additionalcomponents.

U.S. Pat. No. 4,042,684, issued to Kahm, on Aug. 16, 1977, discloses abeverage for supplementing the dietetic requirements of sugar andessential salts in a mammalian body depleted through physical activity.The beverage contains an aqueous solution of fructose, glucose, sodiumchloride, potassium chloride, and free citric acid. The patent teachesto include glucose in the beverage in an amount at least twice that offructose. This patent is incorporated herein by reference in itsentirety, including for its teaching regarding the addition ofadditional components.

U.S. Pat. No. 4,309,417, issued to Staples, on Jan. 5, 1982, describes aprotein fortified isotonic beverage containing sodium ions, potassiumions, chloride ions, phosphate ions, and a sweetener. Most of theelectrolytes needed in the beverage are provided by the whey proteinconcentrate added to the beverage. The osmolarity of the beverage rangesfrom about 140 to about 375 mOs/kg. This patent is incorporated hereinby reference in its entirety, including for its teaching regarding theaddition of additional components.

U.S. Pat. No. 4,312,856, issued to Korduner, et al., on Jan. 26, 1982,discloses a beverage product adapted for rapid replacement of liquid andcarbohydrate in the human body during periods of heavy muscle work. Theproduct is a hypotonic solution that is free of monosaccharide. Itcontains mineral salts, soluble oligosaccharides and/or polysaccharides.This patent is incorporated herein by reference in its entirety,including for its teaching regarding the addition of additionalcomponents.

U.S. Pat. No. 4,322,407, issued to Ko, on Mar. 30, 1982, teaches achemical composition for reconstituting with water to provideelectrolyte drink. The drink consists of sodium, potassium, magnesium,chloride, sulfate, phosphate, citrate, sucrose, dextrose, ascorbic acid,and pyridoxine. This patent is incorporated herein by reference in itsentirety, including for its teaching regarding the addition ofadditional components. U.S. Pat. No. 4,448,770, issued to Epting, Jr.,on May 15, 1984, describes a dietetic beverage adapted for humanconsumption to maintain the balance of body fluids during periods offluid depletion or potassium depletion. The beverage contains potassiumions, calcium ions, magnesium ions, and sucrose. The amount of sucrosepresent ranges from 5 to 10 ounces per gallon of the beverage. Thispatent is incorporated herein by reference in its entirety, includingfor its teaching regarding the addition of additional components.

U.S. Pat. No. 4,551,342, issued to Nakel, et al., on Nov. 5, 1985,describes a beverage suitable for carbonated soft drinks having a pHrange from about 2.5 to about 6.5. The beverage contains a mixture ofcalcium, potassium, and magnesium cations, defined by a first regressionformula. Also included are acids, such as citric, malic, succinic andphosphoric acids, defined by a second regression formula. This patent isincorporated herein by reference in its entirety, including for itsteaching regarding the addition of additional components.

U.S. Pat. No. 4,592,909, issued to Winer, et al., on Jun. 3, 1986,teaches a water based drink formulated for consumption by an athlete.The drink contains water to which have been added salts of sodium,potassium, calcium, and magnesium. The drink does not contain any sugarso that the osmolality of the drink can be kept low. This patent isincorporated herein by reference in its entirety, including for itsteaching regarding the addition of additional components.

U.S. Pat. No. 4,649,051, issued to Gyllang, et al., on Mar. 10, 1987,discloses a beverage product adapted for administration of water andcarbohydrates to a human body. The drink is monosaccharide-free. Thispatent is incorporated herein by reference in its entirety, includingfor its teaching regarding the addition of additional components.

U.S. Pat. No. 4,737,375, issued to Nakel, et al., on Apr. 12, 1988,teaches beverages and beverage concentrates nutritionally supplementedwith mixtures of citric, malic, phosphoric acids, and also significantlevels of solubilized calcium. The beverages and concentrates aresubstantially free of sugar alcohol. This patent is incorporated hereinby reference in its entirety, including for its teaching regarding theaddition of additional components.

U.S. Pat. No. 4,738,856, issued to Clark, on Apr. 19, 1988, teaches abeverage solution containing ions of calcium, magnesium and potassium.The beverage also contains a sweetener and a stabilizer. This patent isincorporated herein by reference in its entirety, including for itsteaching regarding the addition of additional components.

U.S. Pat. No. 4,830,862, issued to Braun, et al., on May 16, 1989,describes beverages and beverage concentrates supplemented withsignificant levels of solubilized calcium and low levels of sulfate andchloride ions. They also contain acids selected from phosphoric acid,citric acid, malic acid, fumaric acid, adipic acid, gluconic acid, andlactic acid, as well as mixtures of these acids. This patent isincorporated herein by reference in its entirety, including for itsteaching regarding the addition of additional components.

U.S. Pat. No. 4,871,550, issued to Millman, on Oct. 3, 1989, teaches anutrient composition. The composition contains free amino acids,carbohydrates, vitamins, minerals and trace elements, electrolytes, andflavoring aids. This patent is incorporated herein by reference in itsentirety, including for its teaching regarding the addition ofadditional components.

Canadian Patent No. 896486, issued to Babagan, et al., on Mar. 28, 1972,teaches an essentially isotonic beverage containing dextrose andelectrolytes in contrast to the customary beverages that utilizesucrose. This patent is incorporated herein by reference in itsentirety, including for its teaching regarding the addition ofadditional components.

A wide variety of electrolyte and sport drinks are available in themarket. These drinks allegedly replenish water, carbohydrates, essentialelectrolytes, and other ingredients lost from a human body throughdehydration.

Gatorade® Thirst Quencher, marketed by Stokely-Van Camp, Inc., containsabout 6% of sucrose and glucose. It also contains sodium, potassium,chloride, and phosphorus. The drink has an osmolality in the range ofbetween 280-360 mOs/liter.

Exceed® Fluid Replacement & Energy Drink, marketed by Ross Laboratories,contains about 7% of glucose polymers and fructose. It also includessodium, potassium, calcium, magnesium, and chloride. The drink has anosmolality of 250 mOs/liter.

Quickkick®, marketed by Cramer Products, Inc., contains about 4.7% offructose and sucrose. The drink is also provided with sodium, potassium,calcium, chloride, and phosphorus. The drink has an osmolality of 305mOs/liter.

Sqwincher® the Activity Drink, marketed by Universal Products, Inc.,contains glucose, fructose, sodium, potassium calcium, magnesium,phosphorus, chloride, and Vitamin C. The drink has an osmolality of 470mOs/liter.

10-K™, marketed by Beverage Products, Inc., contains sucrose, glucose,fructose, sodium, potassium, Vitamin C, chloride, and phosphorus. Thedrink has an osmolality of 350 mOs/liter.

USA Wet™, marketed by Texas Wet, Inc., contains sucrose, sodium,potassium, chloride, and phosphorus. The drink has an osmolality of 450mOs/liter.

Additional patents that disclose adding components to drinks includeU.S. Pat. No. 5,114,723, issued to Stray-Gundersen, on May 19, 1992;U.S. Pat. No. 5,891,888, issued to Strahl, on Apr. 6, 1999; U.S. Pat.No. 6,455,511, issued to Kampinga, on Sep. 24, 2002; U.S. Pat. No.6,989,171, issued to Portman, on Jan. 24, 2006. All of which areincorporated herein by reference in their entirety, including for itsteaching regarding the addition of additional components.

Sports drinks to enhance stamina have been disclosed.

Prinkkila in U.S. Pat. No. 4,853,237 discloses a fitness drink powdercontaining glucose polymer, various salts and fruit acid. The drinkcomposition of Prinkkila is designed to be available to the body in anoptimum manner. In addition, the drink product is designed to maintain ahigh sugar concentration in the blood during physical exertion.

In U.S. Pat. No. 5,032,411 Stray-Gunderson discloses a hypotonicbeverage with essential electrolytes, minerals and carbohydrates.Because the beverage composition is hypotonic, the stomach empties veryrapidly and the composition can produce a beneficial physiologicresponse.

Kahm in U.S. Pat. No. 4,042,684 discloses a dietetic beverage containingsugar and essential salts. The composition is said to enhance energystores. In addition, the composition does not require preservatives. Themixture of glucose and fructose used in the composition produces rapidtransport of glucose out of the digestive system while fructose is moreslowly transported out of the system.

Strahl in U.S. Pat. No. 6,039,987 discloses a composition to preventdehydration and prevent cramps which contains electrolytes,carbohydrates and quinine.

King in U.S. Pat. No. 5,780,094 discloses a sports beverage containing asaccharide in the amount of 1.25% weight to volume of glucose.

Simone in U.S. Pat. No. 5,397,786 discloses a rehydration drink thatcontains carbohydrate, various electrolytes and one ammonia neutralizersuch as aspartate, arginine and glutamate.

A flavored and sweetened aqueous dietetic beverage used to rehydrate thebody is disclosed by Boyle in U.S. Pat. No. 4,874,606.L-aspartyl-L-phenyl-alanine methyl ester is included in the beverage toincrease the degree of gastric emptying.

EXAMPLES Example 1 (According To Particular Aspects, ProducingBiologically Active Electrokinetic Fluids is Enhanced by Processing theFluids as Described Herein at Low Temperatures and/or Elevated GasPressures)

According to particular aspects, the optimal temperature for theelectrokinetic production of the biologically active electrokineticaqueous fluids is at a temperature between about −2° C. and about 10°C., between about −2° C. and about 5° C., between about 0° C. and about5° C., and preferably at about 4° C. or a temperature equivalent to themaximum aqueous density of the fluid being processed (as the maximumdensity may vary somewhat with the salt concentration of the aqueousfluid). In particular aspects, the optimal temperature for theelectrokinetic production of the biologically active electrokineticaqueous fluids is at about 0° C. to about 4° C., 0° C. to about 3° C.,0° C. to about 2° C., 0° C. to about 1° C., 2° C. to about 4° C., or 3°C. to about 4° C.

In additional aspects, the optimal temperature for the electrokineticproduction of the biologically active electrokinetic aqueous fluids isat the temperature of highest density of the aqueous (e.g., water orsaline) fluid (e.g., 4° C.). For pure water this occurs at about 4° C.,and is 1.0000 g/cm³ plus or minus 0.0001.

Without being bound by mechanism, the optimal temperature for producingbiologically active electrokinetic fluids likely coincides withproviding a water structure that unexpectedly (with respect to waterstructures present at other temperatures or temperature ranges)facilitates the formation of charge-stabilized oxygen-containingnanostructures according to the present specification.

As appreciated in the art, the maximum density of water occurs at about4° C., with the density falling with decreasing or increasingtemperature. According to particular aspects, this temperature densityprofile indicates that prior to freezing, there is a unique waterstructure that occurs at or near the maximum density, and/or between thefreezing point temperature of the aqueous fluid and the temperature ofmaximum aqueous density of the fluid, wherein said unique waterstructure optimally facilitates the formation of charge-stabilizedoxygen-containing nanostructures according to the present specification.In particular aspects, this may reflect a water structure providinghydrogen bonding that is similar to, or required in the hydrogen bondingin the aqueous hydration shells of the charge-stabilizedoxygen-containing nanostructures according to the present specification.

In further aspects, optimal production of biologically activeelectrokinetic aqueous fluids comprises introduction of gas at elevatedpressures, including at elevated pressures in combination with theoptimal temperature parameters discussed above. The standard atmosphere(atm) is a unit of pressure defined as being equal to 101,325 Pa or101.325 kPa (e.g., 0.101 Mpa, 760 mmHg (Torr), 29.92 in Hg, 14.696 PSI,1013.25 millibars). In particular aspects, the gas (e.g., oxygen gas inintroduced into the mixing chamber of the electrokinetic mixing deviceat a pressure of at least 0.5 psi, at least 15 psi, at least 30, atleast 45, at least 60, at least 75, at least 90, and preferably apressure is used that is between about 15 psi and 100 psi. In particularaspects the oxygen pressure is at least 35 psi. In particular aspects,there is a gradient of pressure across the rotating mixing chamber fromone end to the other (e.g., from 25 psi to 15 psi, with a 10 psi dropacross the device), where such gradient may comprise, for example,pressure gradients within the range of about 0.5 psi and 100 psi, orwithin a sub range thereof.

In particular aspects, feature-induced cavitations of a rotating mixingdevice having an array of features (US 2008/02190088 (now U.S. Pat. No.7,832,920), US 2008/0281001 (now U.S. Pat. 7,919,534); US 2010/0038244,WO 2008/052143,) provide for elaboration, within the mixing chamber, ofdiscontinuous cavitation-induced pressurization-depressurization eventslocalized at or near the features during electrokinetic fluidprocessing. In particular aspects, elaboration of discontinuouscavitation-induced pressurization-depressurization events with themixing chamber is combined with introduction of oxygen to the mixingchamber at increased pressure as described above.

In particular aspects, oxygen is introduced into the mixing chamber ofthe electrokinetic mixing device by introducing liquid oxygen into themixing chamber of the electrokinetic mixing device.

Example 2 (The Stability of the Biological Activity of The DisclosedElectrokinetic Aqueous Fluids Was Shown to be Temperature Dependent)

RNS60 modulates the expression of IL8. RNS60 is produced when isotonicsaline is processed through the disclosed electrokinetic mixing deviceunder 1 atmosphere of oxygen back-pressure, resulting in an oxygencontent of 60 ppm. In order to test if RNS60 has additional effects onthe bronchial epithelial cells, we tested for the release of multipleinflammatory mediators. Inflammation of the airway tissues is consideredhighly relevant in respiratory disease progression. Respiratory tractproinflammatory cytokines, such as IL-8 are used to access theepithelial cell response to environmental stimulants. In thisexperiment, we tested the effects of RNS60 on IL-8 secretion in humanairway epithelial cells, when challenged with diesel exhaust particle(DEP) and recombinant TNFa (rTNFα).

Methods. The HBEpC cells were pretreated with serum free mediacontaining saline solutions (NS or RNS60) and incubated at 37° C. for 1hour. Cells were then stimulated with rTNFa or DEP and incubated overnight. HBEpC culture supernatants were harvested and IL-8 ELISA wasperformed.

Results. The experiments were repeated three times and representativedata is shown here. FIG. 47a shows that RNS60 regulates DEP-induced IL8secretion.

HBEpC were pre-treated with 10-30% NS or RNS60 for 1 hour, thenstimulated with 100 ug/ml of DEP and incubated overnight at 37° C.,supernatants were harvested and IL-8 ELISA was performed. Averaged datafrom three replicates are shown here.

FIG. 47b shows that RNS60 regulates rTNFa induced IL8, and it retainsits biological activity at room temperature for days. HBEpC werepre-treated with 30% NS or RNS60 and stimulated with 100 ng/ml of rTNFaas described above, supernatants were harvested and IL-8 ELISA wasperformed. Averaged data from three replicates are shown.

As shown in FIGS. 47a & b, RNS60 significantly suppresses DEP or rTNFainduced IL-8 secretion by primary human bronchial cells. Moreover, thebiological activity (IL-8 efficacy) of RNS60 can be reversed, at leastin part, by leaving the fluid at 18-22° C. (room temperature) for 8 to10 days as demonstrated by FIG. 47 b.

According to particular aspects, therefore, once produced, the stabilityof the biological activity of the disclosed electrokinetic aqueousfluids is also temperature dependent. According to particular aspects,the optimal temperature for the stability of the biological activity ofthe disclosed electrokinetic aqueous fluids is at a temperature betweenabout −2° C. and about 10° C., between about −2° C. and about 5° C.,between about 0° C. and about 5° C., and preferably at about 4° C. or atemperature equivalent to the maximum aqueous density of the fluid beingprocessed (as the maximum density may vary somewhat with the saltconcentration of the aqueous fluid). In particular aspects, the optimaltemperature for the stability of the biological activity of thedisclosed electrokinetic aqueous fluids is at about 0° C. to about 4°C., 0° C. to about 3° C., 0° C. to about 2° C., 0° C. to about 1° C., 2°C. to about 4° C., or 3° C. to about 4° C.

In additional aspects, the optimal temperature for the stability of thebiological activity of the disclosed electrokinetic aqueous fluids isalso at the temperature of highest density of the aqueous (e.g., wateror saline) fluid (e.g., 4° C.). For pure water an most saline solutionsthis occurs at about 4° C., and is 1.0000 g/cm³ plus or minus 0.0001.

In particular aspects the stability of the biological activity of theelectrokinetically-altered fluid is also enhanced if it is stored in aclosed container with no, or little “head” volume. Preferably, if thereis a “head volume” in the storage container, it is minimaloxygen-comprising head volume.

Example 3 (Beneficial Effects of Electrokinetically Processed Fluids onHuman Exercise Performance and/or Recovery Were Demonstrated) Overview:

Athletic performance is determined by many parameters that include age,genetics, training, and biomechanics. In addition, optimized diet andhydration are important factors in achieving and sustaining maximalperformance. Beverages currently available in the market place aim atboosting performance by additives that include electrolytes, proteins,carbohydrates, or caffeine. Particular aspects of the present inventionprovide an innovative approach to athletic performance enhancement byoffering a product that protects muscle cells through charge-stabilizednanostructure (CSN) technology.

CSN-containing fluids were generated using Applicant's proprietaryprocess that involves Taylor-Couette-Poiseuille flow in the presence ofoxygen.

The effects of electrokinetically-processed water on human treadmillexercise performance was evaluated by Seattle Performance Medicine,Seattle, Wash.

According to particular aspects, therefore, daily consumption of RSBprior to strenuous exercise improves performance and enhance trainingadaptation.

Materials and Methods:

RSB was produced using a proprietary pump involvingTaylor-Couette-Poiseuille flow in the presence of oxygen as describedherein. The test fluid was an electrokinetically-altered purified water(BEV-A) processed as described herein (see also US2008102190088 (nowU.S. Pat. No. 7,832,920), US2008/0281001 (now U.S. Pat. No. 7,919,534);US201010038244, WO2008/052143). The dissolve oxygen concentration (D.O.)for the test fluid was 52.4 ppm. The control (negative control) was acorresponding, but non-electrokinetically-processed purified water.

A double blind, randomized crossover study, was performed wherein 25fit, male subjects (age: 18 to 35 years) consumed either RSB(CSN-containing fluid; BEV-A) or purified water (PW) as control for 2weeks followed by a 60-minute treadmill exercise at 75% maximal oxygenconsumption (VO₂max). VO₂max was determined according to the ModifiedAstrand protocol and rating of perceived exertion (RPE) was recordedaccording to the Modified Borg Scale at 15 and 50 minutes. Plasmamarkers of skeletal muscle breakdown, myoglobin and creatine kinase(CPK), were measured by ELISA. In addition, plasma concentrations ofselected cytokines were measured with a Luminex 52-plex cytokine assay.

Consent forms, dietary and exercise protocols and diary workbooks wereprovided to all subjects. Prior to consumption of the study test andcontrol beverages, Groups A and B consumed the samenon-electrokinetically-altered “washout” beverage. Group A subjectsbegan to consume their respective washout beverage one day before

Group B and consumption of washout beverage continued for approxone-month prior to the start of the comparative study.

Group A and B subjects consumed their respective alternate beverage for2 weeks.

TABLE 3 Day 1 Day 2 Day 3 Day 4 Day 5 M T W Th F VO₂max 1 VO₂max 1Exhaustive Exhaustive Group A Group B Test Test Group A Group B 10-12subjects 10-12 subjects Schedule in pairs 1.5 hr blocks Schedule inpairs 2 hr blocks  9:00-10:30 7:00-9:00 10:30-12:00  9:00-11:0012:00-1:30  11:00-1:00  2:00-3:30 1:00-3:00 3:30-5:00 3:00-5:005:00-6:30 (optional) 5:00-7:00 (optional)Subjects maintained hydration, carbohydrate intake and exercise levelsduring the study.

Test Article:

Both control and test articles were stored under refrigeratedconditions. Each bottle was labeled at the time of manufacture with thelabel key confidentially maintained at the Sponsor's facility. Both testand control articles were distributed to the subjects and keptrefrigerated prior to consumption.

Study Subjects:

The study subjects were males between the ages of 18 and 35, havingvarying levels of fitness. The subjects were determined to be in goodhealth with no pre-existing conditions and on no medications. Diet andactivity levels were standardized prior to the start of the test articleconsumption.

Blood Samples:

Blood samples were taken for lactic acid at 20, 40 and 60 minutes (justprior to the end of exercise) during exercise. Venous samples were drawnprior to each beverage cycle, just prior to exercise, at 30 and 60minutes after the start of exercise, and then 24 hrs after the exercisewas completed. These samples included a cbc, complete metabolic profile,magnesium, calcium, phosphorous, myoglobin, lactic acid, CPK, CRP andLuminex cytokine analysis. Luminex samples were analyzed for cytokinesincluding IL-1b, TNF-a, IL-6, IL-8, INF-g, IL-4, and others asdetermined by the sponsor. 24-hr urine collection for total volume,creatinine clearance, osmolarity and urine electrolytes was collectedprior to starting beverage, prior to exhaustive exercise and 24 hrsafter exercise.

Statistical Analysis:

Statistical analysis was stratified based on VO₂ Max (as example: 25-40ml/kg/min and 41-60 ml/kg/min) and also examined as a whole.

VO₂ max, or maximal oxygen uptake, is one factor that can determine anathlete's capacity to perform sustained exercise and is linked toaerobic endurance.

VO₂ max refers to the maximum amount of oxygen that an individual canutilize during intense or maximal exercise. It is measured as“milliliters of oxygen used in one minute per kilogram of body weight.”

RPE refers to the rating of perceived exertion, wherein a visual analogscale was used to assess perceived fatigue (i.e., maximal exertion), atthe end of the VO2 max.

Lactate refers to blood Lactate levels. Lactate in the blood can becorrelated with the accumulation level of lactic acid in muscle tissue.

Results:

In Table 4 and FIG. 48, “P” corresponds to control (non-electrokineticbeverage) group, and “R”: corresponds to the electrokinetic beveragetest group.

The results (see Table 4 below and FIG. 48) indicate that the beveragehad an effect on all 3 parameters of exercise performance, and that thedirection of the effect was favorable direction in all 3 areas (positivefor VO₂ max, negative for RPE (rating of perceived exertion) negativefor lactate). The RPE had the most substantial shift, and is the mostrelevant factor for exercise performance.

Documenting performance improvement is really the end point goal of allresearch in sports science. Measurements such as VO2, lactate, your labtests results are mildly persuasive but performance data is what reallymoves athletes, coaches and sports scientists.

TABLE 4 Largest Largest Average Change Change Change Total P R P RVO₂max 2-1 −1.078 −1.826 −0.267 −7.540 −5.380 RPE 2-1 −0.260 0.250−0.813 1.500 −2.000 Lactate 2-1 −0.808 −0.423 −1.225 −2.380 −4.850 MEANSSTANDARD DEVIATIONS T-VALUE vo2max: 8216 3.1191 1.3170 RPE −.5200 1.1248−2.3115 lactate: −.3679 1.9895 −0.9246 T-test conclusions: vo2max: With80% confidence, the expected value of (R − P) will be in [−.0006,1.6438]. With 95% confidence, the expected value of (R − P) will be in[−.4660, 2.1092]. RPE: With 95% confidence, the expected value of (R −P) will be in [−.9843, −.0557]. Note that the expected value of R − P isnegative with 95% confidence. lactate: With 80% confidence, the expectedvalue of (R − P) will be in [−.8923, .1565]. With 95% confidence, theexpected value of (R − P) will be in [−1.189, .4534].

According to particular aspects, therefore, the present invention hassubstantial utility for enhancing exercise performance and recovery.

In particular aspects, the present invention has substantial utility formaintaining, and in some aspects normalizing, a reduced oxygenated bloodlevel in an animal subsequent to a blood oxygen-lowering effectactivity, such as what typically occurs in an animal, such as a human,after an oxygen-consuming activity, such as exercise. Changes in thesephysiologically measurable parameters are typically attendant anincrease in physical activity, stress or other fatigue-inducing event.

In particular aspects, changes in heart rate, oxygen saturation, bloodlactate, oxygen consumption, and fatigue assessment by a patient inresponse to a defined exercise regimen were favorably improved afterconsuming a defined quantity of the electrokinetically-altered fluidsrelative to control non-electrokinetically-altered fluid.

In particular aspects, the present invention has substantial utility forinhibiting and/or delaying onset of fatigue in a human. In particularaspects, subjects consuming electrokinetically-altered oxygenated fluidshave a lesser drop in oxygen saturation compared to subjects consumingelectrokinetically-altered oxygenated fluids.

In particular aspects, the present compositions and methods havesubstantial utility for inhibiting and/or reducing the increase inlevels of blood lactate attendant human exercise, for reducing musclesoreness, and for reducing lactic acid accumulation in muscle.

In particular aspects, the present compositions and methods havesubstantial utility for reducing and/or inhibiting the onset of fatiguein response to exercise in a human.

In particular aspects, the present compositions and methods havesubstantial utility for increasing and/or replenishing available oxygenin the blood stream by consuming the oxygen-enriched nanostructuredfluid preparations.

Example 4 (Consumption of the Disclosed Sports Beverage Altered Markersof Exercise Performance and Cardio-respiratory Fitness) Overview:

Currently available beverages designed to boost performance containadditives that include electrolytes, proteins, carbohydrates, orcaffeine. Disclosed herein is a novel approach providing a beverage thataims at protecting muscle cells through charge-stabilized nanostructures(CSN). CSN-containing solutions are generated through Applicants'proprietary process that involves Taylor-Couette-Poiseuille flow in thepresence of oxygen. Applicants have previously demonstrated that RNS60,a saline therapeutic formulation, alters the cellular response tovarious stressors through effects on voltage-gated ion channels andpotentially other voltage-sensing proteins. Voltage-gated ion channelstightly regulate skeletal muscle contraction and cardiac function, andmaximum oxygen uptake (VO₂max), a widely used measure ofcardio-respiratory fitness, is in part determined by cardiac output.

In this Example, Applicants investigated whether oral consumption ofApplicants' Sports Beverage (RB), a water beverage processed in asimilar manner to RNS60, would alter selected physiological responsesduring exercise. In a double blind, randomized, crossover study, RBconsumption led to a 5% increase in VO₂max in highly fit subjects and adecrease in the rating of perceived exertion in lesser-trained subjects.In addition, RB lowered the plasma levels of myoglobin and creatinekinase, two markers of the response to strenuous exercise, andattenuated exercise-induced circulating levels of several cytokines.Test and control fluids were as described in Example 3.

According to particular aspects, therefore, ingesting RB (e.g., in dayspreceding strenuous exercise) improves performance and enhances trainingadaptation.

Athletic performance is determined by many parameters that include age,genetics, training, and biomechanics. In addition, optimized diet andhydration are important factors in achieving and sustaining maximalperformance. Beverages currently available in the market place aim atboosting performance by additives that include electrolytes, proteins,carbohydrates, or caffeine. Applicants have developed an innovativeapproach to athletic performance enhancement by providing fluidcompositions that protect muscle cells through charge-stabilizednanostructure (CSN) technology.

As primary study endpoints, Applicants measured maximum oxygen uptake(VO₂max) and rating of perceived exertion (RPE). VO₂max has been linkedto aerobic exercise performance, and while it is clear that VO₂max aloneis not a predictor of overall athletic performance, it is an importantmeasure of cardio-respiratory fitness [2]. VO₂max is influenced to alarge extent by cardiac output, and cardiac function is controlled by atightly regulated interplay of voltage-gated ion channels [3]. RPE is asubjective rating of physical exertion that is widely used as a generalmeasure of physiological stress and the capability to sustain physicalexercise [4]. In addition, Applicants measured plasma levels ofmyoglobin and plasma creatine kinase (CK), two routinely used markers ofskeletal muscle damage [5].

Strenuous exercise has been shown to induce a cytokine response[6].Among the cytokines released from skeletal muscle, interleukin-6 (IL-6)has been reported to undergo the most rapid and profound upregulation[6,7,8]. IL-6 has been suggested to induce an anti-inflammatory responsethat may be involved in preventing excessive tissue damage [9], but ithas also been linked to general fatigue and underperformance syndrome(a.k.a. overtraining syndrome), a syndrome that is characterized bysymptoms ranging from lack of performance improvement to signs ofclinical depression [10,11]. In two independent treadmill exercisestudies, plasma levels of IL-6 have been linked to VO₂max and RPE: inthe first study, IL-6 levels were inversely correlated with VO₂max [12],whereas in the second, the lowering of plasma IL-6 levels induced byconsumption of a carbohydrate beverage was associated with a lower RPE[13].

The response of other cytokines to exercise is less well studied.Circulating levels of soluble CD40 ligand (sCD40L, CD154) have beenreported to be lowered by ultra-endurance exercise in athletes [14] andmoderate exercise in heart failure patients [15]. CD40 and its ligandare involved in inflammatory processes in atherosclerotic plaque and thedevelopment of arterial thrombi that cause myocardial infarction, andsCD40L was identified as a risk marker in patients with acute coronarysyndrome [16]. Colony-stimulating factors including macrophagecolony-stimulating factor (M-CSF) have been shown to be upregulated inresponse to exercise [17], but the implications are unknown at thistime. Based on the increasing recognition of exercise effects related toinflammatory processes, Applicants screened for changes in circulatinglevels of a comprehensive panel of cytokines using a Luminex 52-plexcytokine assay.

Study Design

Study participants were a blend of performance-oriented individuals andthe general health-conscious population. Diet and activity levels werestandardized prior to the testing, and subjects were instructed toconsume a consistent diet throughout the entire study. For the 24-hourperiod preceding exercise testing, subjects were instructed to consumethe same menu consisting of ˜60% carbohydrate, ˜15% protein and ˜25%fat. Subjects were allowed to drink and eat freely until theirpre-exercise venous blood sampling was obtained; from that point on theyconsumed approximately 10 ounces plain water until beginning theexercise, and they did not consume anything during the 60-minuteendurance sessions. Subjects were advised to refrain from long or hardworkouts for 2 days prior to their VO₂max test and throughout the trialweeks. They were also instructed to refrain from altering their trainingunless required to by the study design.

The study was designed as a crossover study (FIG. 49). Study subjectswere randomized to two groups. One group consumed 1.5 L of RB per dayfor 2 weeks, and after a washout period of 2 weeks consumed 1.5 L perday of placebo water (PW). The other group started with consumption of1.5 L of PW for 2 weeks, and after the washout period continued with 1.5L per day of RB.

FIG. 49 shows a study design overview. Study subjects were randomized to2 groups. Group 1 consumed 1.5 L of RB per day for 2 weeks, and after awashout period of 2 weeks consumed 1.5 L per day of PW. Group 2 startedwith consumption of 1.5 L of PW for 2 weeks, and after the washoutperiod continued with 1.5 L per day of RB. Within each of the twobeverage consumption periods, VO₂max was determined on day 12 and anexercise testing was performed on day 15.

VO₂max measurements. On day 12 of beverage consumption, VO₂max wasdetermined according to the Modified Astrand protocol (variable 5-8 mphand increasing by 2.5% incline per stage). Subjects who required greaterthan 8 mph velocity used the Costill and Fox protocol (8-9 mph andincreasing by 2% incline per stage). All tests were completed on PrecorTreadmills under standard laboratory conditions of temperature (62-67°F.), pressure (700-715 mmHg) and relative humidity (30-40%). Prior toeach test, the equipment was calibrated. The exercise protocol requiredsubjects to complete a 10-minute self-paced warm-up before beginning thefirst stage of the test, which targeted an RPE of 11. Following a3-minute first stage, the incline was increased every 2 minutes to thepoint of maximal effort. On completion of the test, final power outputand VO₂max were noted. 75% of the VO₂max power output was calculated asa target for the exercise testing, which followed 3 days later.

Exercise testing. Exercise testing was conducted on day 15 of beverageconsumption. During each test, subjects ran continuously for 60 minutesat 75% VO₂max on a level treadmill. Participants performed bothexercises on the same treadmill and at the same pace. Blood samples werecollected from the antecubital veins at three time intervals:immediately pre-exercise, 30 minutes into the exercise, just prior tocompletion of the exercise at 60 minutes, and 24 hours after completionof the exercise. For the sampling during the exercise, subjects slowedto a walk 3.0-3.5 mph for less than 3 minutes (average: 2.5 minutes)before returning to their running pace. The blood samples were used tomeasure complete blood count, complete metabolic profile, magnesium,calcium, phosphorous, myoglobin, lactic acid, creatine kinase (CK), andC-reactive protein (CRP). In addition, the plasma concentrations of apanel of cytokines were measured with a Luminex 52-plex cytokine assay.

RPE was recorded according to the Modified Borg Scale at 15 and 50minutes. Lactate measurements were obtained utilizing an AccusportLactate Plus analyzer at the following times: pre-exercise, duringexercise at 20, 30, and 40 minutes and at the conclusion of exercise at60 minutes. Twenty four-hour urine collection for total volume,creatinine clearance, osmolarity, and urine electrolytes were collectedprior to starting beverage, prior to exhaustive exercise and 24 hoursafter the exercise.

Results and Discussion:

RB consumption improved VO₂max in experienced athletes. Twenty-five fitmale subjects (age: 18-35 years) were randomly assigned to the studygroups. The study groups comprised individuals with a regular traininghistory as well as more sedentary individuals, with a mean VO₂max of53.4 mL/kg/min. A comparison of VO₂max values within the entire studypopulation did not reveal a difference between subjects receivingApplicants' Sports Beverage (RB) and subjects receiving normal, purifiedwater (PW). However, when study subjects were analyzed based on theirbeginning fitness level, a 5% improvement in the subgroup with a VO₂maxabove 60 mL/kg/min was observed (FIG. 50A). The difference did not reachstatistical significance, likely due to the small number of subjects inthis VO₂max range (n=6). It is noteworthy, though, that 5 out of the 6subjects showed an increase in VO₂max (FIG. 2B). In study subjects witha VO₂max below 60 mL/kg/min, there was no difference in VO₂max betweenthose who consumed RB and those who consumed PW (FIG. 2A).

FIGS. 50A and B show that RB consumption improves VO₂max in fitterathletes. FIG. 50A. Data are presented as percent change in RB groupsseparated by a VO₂max threshold of 60, compared to the corresponding PBgroups (mean±SEM). FIG. 50B. Absolute VO₂max values for the six studysubjects with a VO₂max>60 m L/kg/min .

VO₂max is defined by the Fick equation, VO₂max=Q(CaO₂−CvO₂), where Q iscardiac output, CaO₂ is the arterial oxygen content, and CvO₂ is thevenous oxygen content. VO₂max, therefore, is to a large part dependenton cardiac output. In particular aspects, RB has the capacity todirectly or indirectly alter cardiac ion channels. Other elements thatinfluence VO₂max are related to oxygen delivery, uptake, and utilization[2]. Pulmonary contribution is not usually a limiting factor; however,in some elite athletes, exercise-induced hypoxemia (EIH) can result fromvery high stroke volume and rapid pulmonary circulatory transit time,where hemoglobin does not pick up adequate oxygen due to insufficienttime spent at the alveolar level [18,19]. Additionally, theoxygen-carrying capacity of the blood, which is determined by plasmavolume, iron status, and hemoglobin levels, is an important component ofoxygen delivery [20]. At the muscle level, capillary density andmembrane diffusion contribute to oxygen delivery, and mitochondrialdensity as well as enzyme and substrate status determine oxygenutilization rate [20].

While VO₂max is a trainable exercise parameter, it has been shown tohave a genetic component, and among adults, high and low responders havebeen reported [21]. A 4.1% VO₂max increase was reported for a group ofelite athletes after an intensive 24-day “live high-train low” altitudetraining regimen [22], and in untrained subjects, training at 75% ofaerobic power for 30 minutes, 3 times a week, over 6 months, wasnecessary to yield an average VO₂max increase of 15-20% [23]. The 5%increase observed in the fitter athletes of our study represents asubstantial improvement considering the short period of beverageconsumption and the absence of a rigorous training program.

While a high VO₂max is required for competitive exercise performance, itis not the only determinant of the actual exercise performance. As anadditional exercise response parameter, the rating of perceived exertion(RPE) was measured.

RB consumption altered RPE. RPE was measured at time points of 15 and 50minutes during the treadmill exercise. As expected, RPE increased overtime (FIG. 51). After consumption of RB, the study participants showed alower RPE when compared to PW consumption (FIG. 3); this wasparticularly apparent in the subgroup of participants with a VO₂max<60mL/kg/min (FIG. 3). The more highly trained subgroup likely operated ona lower RPE level compared to the lesser trained subgroup (12.5±1.5 vs.14.8±0.3, p=0.02) and therefore may not have benefitted as much from theeffect of RB.

FIG. 51 shows that RB consumption decreases RPE. RPE was recordedutilizing a Modified Borg Scale at 15 and 50 minutes during theperformance exercise. The data are presented separated by aVO₂max-threshold of 60 mg/kg/min.

A lowered RPE means that an individual can complete a given exercisewith a lower level of perceived exertion, or exercise longer untilexhaustion is reached, and therefore indicates a potentially beneficialtraining response.

Crossover study artifacts. When analyzing the data from the secondexercise trial, Applicants noticed that the subjects in the PW group ofthis trial (group 1 in FIG. 49) behaved more like they had in the firsttrial, when they consumed RB, than the PW group of the first trial (datanot shown). This phenomenon was independent of the subjects' VO₂max. Anadaptation to training as the underlying reason is not likely in such ashort time frame. One possible explanation is that the washout periodbetween the two trials was not sufficient to “reset” changes introducedby previous RB consumption. To avoid skewed data based on aninsufficient washout, we excluded study group 1 (RB first) from furtheranalysis and instead analyzed group 2 (PW first) only. All data shownbelow therefore represent the paired analysis of group 2 individualsconsuming PW in trial 1 and RB in trial 2.

RB consumption lowered plasma myoglobin and CK levels. When the studysubjects consumed PW, circulating myoglobin levels increased duringexercise, peaking at a 2.8-fold elevation (absolute mean increase: 73ng/mL) by the end of the exercise. When the subjects consumed RB,however, myoglobin levels did not rise above pre-exercise values (FIG.52). CK levels increased more slowly when compared to myoglobin, whichis in agreement with published reports [5]. The highest increase inplasma CK (1.9-fold, absolute mean increase: 202 units/L) was present 24hours after the endurance trial when the subjects had consumed PW. RBconsumption attenuated the elevation of plasma CK levels at the60-minute and 24h-hour time points in a statistically significant manner(FIG. 52).

FIG. 52 shows time point differences in levels of plasma myoglobin. Dataare presented as differences (mean±SEM) between two time points asindicated by the labels of the x-axis. D0=day before start of beverageconsumption, PE=time point immediately prior to starting the enduranceexercise, 30 min=30-minute time point of endurance exercise , 60min=60-minute time point of endurance exercise (end of exercise), 24h=24 hours after completion of the exercise. P-values were calculated byWilcoxon signed rank test.

FIG. 53 shows time point differences in plasma CK levels. Data arepresented as differences (mean±SEM) between two time points as indicatedby the labels of the x-axis. D0=day before start of beverageconsumption, PE=time point immediately prior to starting the enduranceexercise, 30 min=30-minute time point of endurance exercise, 60min=60-minute time point of endurance exercise (end of exercise), 24h=24 hours after completion of the exercise. P-values were calculated byWilcoxon signed rank test.

According to particular aspects, therefore, the reduction of plasmamyoglobin and CK levels indicates that muscle damage after exercise isattenuated by drinking RB.

Effects of RB Consumption on Plasma Cytokine Levels.

IL-6. Across all study groups, circulating levels of IL-6 were low, witha modest maximal increase of ˜2-fold, from 3.97±7.79 (SD) pg/mL beforethe exercise to 8.30±8.14 (SD) pg/mL by the end of the exercise(p<0.0001). While others have reported post-exercise IL-6 levels toincrease up to 100-fold [24], higher exercise levels and/or longerexercise durations are typically needed to achieve an increase of thismagnitude. In several published studies, for example, running for aperiod of 2-3 hours was associated with maximal IL-6 levels in the rangeof 40 pg/mL to 120 pg/mL [7,13,25].

Applicants did not measure significant differences in plasma IL-6 levelsbetween consumption of RB and consumption of PW (data not shown).Consumption of a 6% carbohydrate beverage was reported by others todecrease circulating IL-6 levels in marathon runners [13]; compared toApplicants' study, however, this effect was observed after a longerexercise duration (2.5 hours) and with IL-6 levels in a higherconcentration range (50-75 pg/mL) [13]. Both, the increase of IL-6 afterprolonged exercise and the lowering achieved by consumption of acarbohydrate-containing beverage, are in agreement with the fact thatIL-6 production is induced in glycogen-depleted skeletal muscle [26,27].

IFN-α, ENA-78, and M-CSF. Two pro-inflammatory cytokines that displayedreduced plasma concentrations when study subjects consumed RB wereinterferon-a (IFN-a) and epithelial neutrophil activating protein 78(ENA-78) (FIG. 54).

FIG. 54 shows that RB inhibited the exercise-induced increase of plasmalevels of IFN-α (A) and ENA-78 (B). Data are presented as differences(mean±SEM) between two time points as indicated by the labels of thex-axis. D0=day before start of beverage consumption, PE=time pointimmediately prior to starting the endurance exercise, 30 min=30-minutetime point of endurance exercise, 60 min=60-minute time point ofendurance exercise (end of exercise), 24 h=24 hours after completion ofthe exercise. P-values were calculated by Wilcoxon signed rank test.

Both cytokines increased 24 hours after completion of the exercise trialthat was performed after 2 weeks of PW consumption; however, thisincrease was absent when the subjects had consumed RB (FIG. 6). IFN-α isa cytokine with a broad range of anti-proliferative activities [28].Similar to IL-6, elevated levels of IFN-α have been linked to fatigueand depression [29,30]. Because IFN-α induces IL-6 [31], these effectsof IFN-α may in part be caused indirectly. ENA-78 is a chemokine of theIL-8 family that has similar effects to the ubiquitous IL-8: it attractsneutrophils and thereby promotes inflammation [32]. Elevated ENA-78levels are found in blood and synovial fluid of rheumatoid arthritispatients and have been associated with disease progression in Crohn'sdisease, ulcerative colitis, acute appendicitis and chronic pancreatitis[32]. ENA-78 expression has been shown to increase in injured skeletalmuscle [33], and the inhibition of plasma ENA-78 by RB therefore isconsistent with the protective effective suggested by the myoglobin andCK data.

M-CSF, IFN-α, and ENA-78 showed the same trend: increased plasma levelsin the subjects consuming PW at the 24-hour time point that were bluntedin subjects receiving RB (data not shown). Absolute M-CSF levels,however, were below the limit of detection in a high proportion ofsamples.

BDNF. Plasma levels of brain-derived neurotrophic factor (BDNF) weremarkedly increased 24 hours after the completion of the exercise trialwhen the study subjects had consumed PW (FIG. 6). RB consumptionabolished the post-exercise BDNF peak (FIG. 55).

FIG. 55 shows that RB consumption prevents the rise in BDNF plasmaconcentration 24 hours after the exercise trial. Data are presented asdifferences (mean±SEM) between two time points, as indicated by thelabels of the x-axis. D0=day before start of beverage consumption,PE=time point immediately prior to starting the endurance exercise, 30min=30-minute time point of endurance exercise, 60 min=60-minute timepoint of endurance exercise (end of exercise), 24 h=24 hours aftercompletion of the exercise. P-values was calculated by Wilcoxon signedrank test.

BDNF is a member of the neurotrophin family that is expressed at highlevels in the brain and in the peripheral nervous system [34]. BDNF hasdiverse effects on neuronal cells, including growth, differentiation,and repair [34], and has been suggested to be a pro-angiogenic factor inischemic tissue [35]. BDNF has also been reported to act as a naturalanti-depressant [36,37] and has been associated with the beneficialeffects of exercise on memory function and depression [38,39]. Incontrast to these positive effects, high BDNF expression in coronaryarteries has been found in patients with heart disease and has beensuggested to contribute to plaque instability and unstable angina [40].How to interpret the reduction of BDNF after RB consumption in our studyis not clear at present. Of note, however, the study subjects with thehighest plasma concentrations of BDNF also had the highestconcentrations of ENA-78 (data not shown). One might speculate thatpost-exercise increases in BDNF, could be linked to the increase ofpro-inflammatory factors such as ENA-78 in a similar fashion to the onethat has been hypothesized for IL-6, TNF-α and IL-1β [9]. In otherwords, the presence of factors that counteract a damage response may notbe needed if the damaging factors are not released in the first place.

sCD40L. In contrast to IFN-α, ENA-78, and BDNF, all of which showedincreased plasma levels 24 hours after exercise, sCD40L showed adifference at earlier time points that was lost later on (FIG. 56).While sCD40L levels increased from the pre-exercise time point to 30minutes into the exercise when the subjects consumed PW, they decreasedwhen the subjects drank RB (FIG. 56). Absolute plasma concentrations,however, were not significantly different between both experimentalconditions (data not shown).

FIG. 56 shows the effect of RB consumption on circulating sCD40L levels.Data are presented as differences (mean±SEM) between two time points, asindicated by the labels of the x-axis. D0=day before start of beverageconsumption, PE=time point immediately prior to starting the enduranceexercise, 30 min=30-minute time point of endurance exercise, 60min=60-minute time point of endurance exercise (end of exercise), 24h=24 hours after completion of the exercise. P-values were calculated byWilcoxon signed rank test.

In summary of this Example. According to particular aspects, Applicants'Sports Beverage (RB) favorably alters the response to exercise. Thestudy demonstrated promising trends caused by RB consumption. Mostimportantly, VO₂max appeared to be elevated in highly fit subjects,whereas lesser-trained subjects showed a trend towards a lower RPE. Atthe same time, plasma myoglobin and CK, two markers of muscle damage,were lower across both study subgroups.

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Example 5 (Beneficial Effects of Electrokinetically Processed Fluids onHuman Exercise Performance and/or Recovery Following Intense Exercise)Overview:

Exercise-induce muscle damage in humans is widely recognized in the art(see, e.g., Clarkson & Hubal, Am. J. Phys. Med. Rehabil. 81:S52-S69,incorporated by reference herein for its teachings on exercise-inducemuscle damage).

According to particular aspects, a blinded (e.g., double-blind,randomized, placebo-controlled, two-arm trial) exercise performance andrecovery test is conducted (e.g., to test the effects on extent ofmuscle fiber micro-injury and recovery), usingelectrokinetically-altered purified water (test fluid), characterizingthat the disclosed electrokinetically-processed fluids have substantialutility for preventing tissue damage and/or enhancing tissue and/orphysiological recovery after intense exercise, including preventingmuscle and/or tendon damage and/or enhancing/facilitating muscle and/ortendon recovery from exercise (e.g., eccentric exercise), particularlyfrom intense exercise.

Materials:

Electrokinetically-altered purified water (test fluid) processed aspreviously described (see also US 2008/0219088 (now U.S. Pat. No.7,832,920), US 2008/0281001 (now U.S. Pat. No. 7,919,534); US2010/0038244, WO 2008/052143, incorporated by reference herein in theirentirety for their respective teachings on processing and properties ofelectrokinetically-altered fluids) is used. The control (negativecontrol fluid) is the corresponding non-electrokinetically-processedpurified water.

Preferred route of administration is oral.

Methods:

An art-recognized arm-curl model (see, e.g., Borsa & Sauers, Med SciSports Exerc 32(5):891-896, 2000; and Borsa & Liggett J Athl Train33(2):150-155, 1998; see also McHugh, et al., Sports Med. 27:157-170,1999, all incorporated herein by reference for their teachings oneccentric exercise models and related measurements) comprising eccentricexercise to the bicep brachii muscle, is used for inducing skeletalmuscle soreness and dysfunction in human subjects using eccentricexercise. The extent of muscle damage and recovery (e.g., the effects onextent of muscle fiber micro-injury and recovery) is determined byanalysis of serum biological markers of muscle damage and/or recovery.In particular aspects, the exercise comprises a single bout of enhancedeccentric exercise.

In certain aspects, muscle microinjury is measured. For, example, aconcentric/eccentric isokinetic exercise protocol for the biceps brachiimuscle is used to induce muscle microinjury (e.g., extent of musclefiber micro-injury and recovery). As a result of this exercise, subjectswill display similar signs and symptoms associated with a mild,sport-related musculotendinous injury. In certain aspects, exercise isperformed on a Kin-Com 500-H dynamometer (Chattecx Corp.). The subject(e.g., male and female adults between the ages of 18-35 years areincluded if they are healthy, non-smoking, and free of nutritional ordietary supplements for a minimum of six weeks) is seated and stabilizedthe same as for typical force measurements. In certain aspects, theangular velocity is set at 30° s⁻¹ for concentric actions, and 60° s⁻¹for eccentric actions. In particular aspects, the range of motion forthe exercise is preset at 45-110° of elbow flexion. Subjects are seatedand the elbow aligned with the axis of rotation of the dynamometer.Subjects typically use their nondominant arm for testing. Each subjectperforms near-maximal concentric and eccentric actions consisting of 10sets of 5 repetitions with a 30-s recovery period between sets.

In certain aspects, peak force production is measured, for example,using a Kin-Com 500-H dynamometer (Chattecx Corp., Chattanooga, Tenn.).Peak force production is the maximal voluntary isometric force producedduring a muscle action. Subjects are typically seated with theirnondominant arm placed at their side in 90° elbow flexion, neutralrotation of humerus, and supination of the forearm. In certain aspects,each subject performs a plurality (e.g., three) of maximal voluntaryisometric actions held, for example, for 2.5 seconds. The average of thevalues is recorded as peak force in Newtons (N). The mid-range positionis used as the reference angle because of the length-tensionrelationship. The length-tension relationship is recognized asdemonstrating that maximal tension is generated at the mid-range ofelbow joint flexion due to optimal available sarcomere cross-bridging.Test/retest reliability is demonstrated.

According to additional aspects, the peak rate of force production isthe steepest point on the slope of the force-time curve, and representsthe muscle's ability to rapidly generate force or tension (N s⁻¹). Tocalculate this value, raw data from the force-time curve is typicallyreduced and displayed using an executable program (e.g., Visual Basic4.0), and test/retest reliability is demonstrated.

In certain aspects, pain perception and muscle dysfunction are assessedas indicators of microinjury. Pain perception is, for example, assessedusing an art-recognized visual analog scale (VAS). The use of a VAS isknown in the art to be a reliable and valid method of quantifying painperception. In certain aspects, the VAS consists of a horizontal line(e.g., 10 cm in length) with 0 at the extreme left representing “nopain” and 10 cm on the extreme right representing “pain as bad as itpossibly could be” for the biceps brachii muscle. Each subject is thenasked to draw a vertical line at the point that most accuratelycorresponds to their perceived level of pain with active flexion andextension of their involved arm.

In certain aspects, pain-free active range of motion (ROM) is used as ameasure of dysfunction. A standard, plastic goniometer may be used toassess ROM for elbow flexion and extension. The goniometer approximatesthe axis of rotation for the ulnohumeral joint and bisects the humerusand forearm. Extension is typically measured with the subject seated andtheir arm resting pain free at their side). For flexion, subjects areasked to flex their elbow to the point just before discomfort. Thisprocess is repeated, for example, twice for both flexion and extensionand the average score is recorded in degrees(°). The criterion measurefor pain-free ROM is calculated by subtracting the extension score fromthe flexion score. Both measures are recorded and analyzed separatelybefore and after the exercise induced microinjury protocol.

For statistical treatment, pre- and post-injury data for pain perceptionand dysfunction are analyzed, for example, using a one-way ANOVA withrepeated measures. Intraclass correlation coefficients (ICCs) andstandard error of measurements (SEMS) are calculated for the forcemeasures. Repeated measures are performed for each dependent variableand the ICC is obtained from analysis of variance taking into accountthe between-subjects mean square, error mean square, trial mean square,number of trials, and number of subjects.

Exemplary detailed study design. A double-blind, randomized,placebo-controlled, two-arm trial is designed to investigate theprotective effects of the test beverage (test bev.) compared tocontrol/placebo (non electrokinetically processed) beverage on andmuscle function following a single bout of enhanced eccentric exercise.Subjects (e.g., male and female adults between the ages of 18-35 yearsare included if they are healthy, non-smoking, and free of nutritionalor dietary supplements for a minimum of six weeks)are randomly assignedto either a test beverage group (n=20) or placebo control group (n=20).The effects of the test bev. are compared to the placebo control.Subjects are required to complete a 21-day supplement trial spanningpre-supplement baseline testing, a controlled water beveragesupplementation regimen, pre-exercise baseline testing, eccentric armexercise to induce muscle damage, and planned follow-up post-exercisedata collection time points. A “between subjects two-arm trial design”is preferably chosen over a “within-subjects cross-over design” in orderto prevent the influence of confounds due to repeated bout effects. Therepeated bout effect is a phenomenon characterized by less muscledamage, inflammation, delayed-onset muscle soreness (DOMS) and strengthdeficits after the second of 2 separate eccentric exercise boutsperformed in close proximity usually within days to weeks.

Study population. Inclusion/Exclusion criteria: male and female adultsbetween the ages of 18-35 years are included if they are healthy,non-smoking, and free of nutritional or dietary supplements for aminimum of six weeks. Supplements include but are not limited toephedra, yohimbine, pro-hormones, creatine or anabolics.

Subjects are excluded if they have been involved in a regularweight-training program within the last six weeks, have a prior historyof injury to the neck, shoulder or elbow region of the non-dominant arm,recent history of a bacterial infection or current reported use ofanti-inflammatory medication within the last 6 weeks.

Test article and dosing. The test beverage and placebo are packaged in500-ml plastic bottles, and subjects are instructed to consume aprescribed dose of the beverage (bottles/day) based on the followingbody weight categories. For example, <130 lb=2 bottles/day; 130-160 lb=3b/d; 160-190 lb=4 b/d; 190-220 lb=5 b/d; >220 lb=6 b/d.

Blood biomarker measures. Assays are used to track serum levels forbiomarkers of muscle damage (e.g., creatine kinase (CK)]) andinflammation (C-reactive protein (CRP), interleukin-6 (IL-6), tumornecrosis factor alpha (TNF-α)) and/or other cytokines or markers asappropriate.

Functional measures. Isometric muscle strength (peak force production)is used as a measure of overall arm function. Symptoms and impairmentsare measured using pressure pain ratings (algometer) and self-reportfunctional questionnaires.

Blood collection. Blood samples are collected via venipuncture in theearly morning (between 7-10 am) after a 10-hr fast. Phlebotomy-trainedpersonnel complete all blood draws. After separation, all specimens arealiquoted into the appropriate number of cryovials based on the proposednumber of assays. Blood is taken from the antecubital vein and collectedinto two 10 mL Vacutainer® tubes containing ethylenediaminetetraaceticacid (K₃EDTA; 8.4 mgNacutainer®) or into two 10 mL serum collectiontubes. Since various biochemical assays require different bloodcollection procedures, two 10 mL Vacutainer® containing either sodiumcitrate (3.2%, 0.109M) and sodium heparin are collected. Blood iscentrifuged at 4° C. at 1500×g for five minutes. For some analyses,plasma is allocated to storage tubes containing 100 μM butylatedhydroxytoluene (BHT), Trolox (100 μM) and 100 μM diethylenetriaminepentaacetic acid (DTPA). DTPA serves as a metal chelator, and BHT actsas a chain-breaking antioxidant to prevent lipid peroxidation ex vivo.Samples are stored immediately at −80° C. in multiple aliquots (6 to 8).Samples stored in multiple aliquots (˜0.25 mL) are thawed only once andimmediately analyzed for a specific biomarker (samples subjected to onefreeze-thaw cycle, may show increases in baseline lipid peroxidationproducts). Cryovial labels are color coded by study and include thesubject ID number, date, visit number (specific per study), and cryovialnumber. Each sample is logged into an Excel spreadsheet by a labtechnician and frozen at −80° C. in locked freezers connected to anemergency power supply and an alarm system.

Visit 1 (Baseline measurements, day 0). Report to lab at assignedmeeting time. A signed informed consent is obtained prior to anytesting. The following procedures are performed: brief medical screen(height, weight, pulse, blood pressure and body temperature), blooddraw, subjective evaluation of arm function, muscle point tenderness,and isometric strength measures. Body temperature is measured to ensurethat the subject is not running a fever. Subjects are instructed tomaintain current activity levels and not to initiate aresistance-training or weight-loss program for the duration of thestudy. Subjects are randomly assigned to a group (test beverage/placebo)and instructed to consume the test beverage (or placebo), which isdispensed for the next 18 days. A pre-exercise visit is scheduled for 10days later. All pre-exercise and follow-up measurements are comparedwith baseline measures.

Visit 2 (pre-exercise measurements, eccentric exercise protocol, day10). Report to lab at assigned meeting time. The following proceduresare performed: brief medical screen (height, weight, pulse, bloodpressure and body temperature), blood draw, subjective evaluation of armfunction, muscle point tenderness, and isometric strength measures.Subjects then undergo a standardized single bout of eccentric resistanceexercise to the non-dominant biceps brachii muscle. A standard arm-curlmachine (Cybex International, Inc., Medway, Mass.) is used for theeccentric exercise protocol. Exercise weights for each subject redetermined by establishing their one repetition maximum (1-RM)concentric or shortening contraction. The 1-RM is determined by havingthe subject perform a concentric arm curl against a low resistance withcontinued repetitions being performed with weight being incrementallyadded until a 1-RM is reached. Each subject then performs five sets often repetitions of lengthening contractions on the arm curl machineusing a weight equivalent to 140% of their 1-RM. Each repetition takes4-6 seconds to ensure maximal tension is being applied to the muscle.Subjects are given a one-minute rest period between sets. Exercise isnot performed in the usual way (concentric)—meaning shortening ofmuscles during contraction. It is rather performedeccentrically—lengthening the muscle while still developing tension(overload). The angular velocity will be set at 45 °/sec for concentricand 60° /sec for eccentric actions. Subjects will be given a one-minuterest period between sets. The exercise session takes less than 10minutes.

Visits 3-5 are scheduled for follow-up measurements.

Precautionary measures are taken to prevent and monitor adversereactions to the exercise protocol. Follow-up visits (e.g., day 19, 20,and 21) are scheduled post-exercise.

Data analysis. Data are analyzed using a two way ANOVA with repeatedmeasures on the second factor (time). Outcome measures are analyzedusing a two (group) by four (time) ANOVA. Statistical significance isset at p<0.05. If significant interactions occur, the Tukey post-hoctest is used to reveal where the differences occur. All data analysesare performed using SPSS^(→) for Windows 16.0 (SPSS, Inc., Chicago,Ill.).

Results:

According to particular aspects, the electrokinetically-processed fluidshave substantial utility for preventing or alleviating/reducing tissuedamage and/or enhancing tissue and/or physiological recovery afterintense exercise, including preventing muscle damage and/orenhancing/facilitating muscle recovery from exercise (e.g., eccentricexercise), particularly from intense exercise.

In certain embodiments, the electrokinetically-processed fluids havesubstantial utility for preventing or alleviating/reducing the extent ofmuscle fiber micro-injury, and/or enhancing recovery thereof.

According to particular aspects, the electrokinetically-processed fluidshave substantial utility for reducing biomarkers of exercise-inducedmuscle injury (e.g., creatine kinase (CK)).

According to additional aspects, the electrokinetically-processed fluidshave substantial utility for reducing subjective ratings of musclesoreness.

According to yet additional aspects, the electrokinetically-processedfluids have substantial utility for preserving muscle contractilefunction (e.g., maximal force, joint ROM).

According to yet additional aspects, the electrokinetically-processedfluids have substantial utility for preventing and/orameliorating/reducing exercise-induced tendon damage and/orenhancing/facilitating tendon recovery from exercise and/orexercise-induced damage (e.g., eccentric exercise, tendinosis,tendonitis, tenosynovitis), particularly from intense exercise, and/orreducing exercise-induced tendon-related pain and/or swelling. Incertain aspects at least one of peroneal tendon, flexor tendon, Achillestendon is treated.

According to yet additional aspects, the electrokinetically-processedfluids have substantial utility for preventing and/orameliorating/reducing and/or enhancing recovery from tendon strainassociated with chronic, repetitive movement, comprising administration,to a subject in need thereof, a electrokinetically-altered sportsbeverage composition as disclosed herein in an amount sufficient toprevent and/or ameliorate and/or enhance recovery from tendon strainassociated with chronic, repetitive movement.

According to further aspects, and as demonstrated in Example 3 above andin this Example 4, the electrokinetically-processed fluids havesubstantial utility for improving exercise performance.

1. A method for enhancing exercise performance and/or recovery time,comprising administering to a subject in need thereof, an oxygenatedionic aqueous solution of charge-stabilized oxygen-containingnanobubbles having an average diameter of less than 100 nanometers in anamount sufficient to provide for enhancing exercise performance and/orrecovery time, wherein ionic aqueous solution comprises oxygen in anamount of at least 15 ppm at ambient temperature and atmosphericpressure, wherein the exercise comprises at least one of intenseexercise, eccentric exercise, exercise in elevated ambient temperature,repetitive exercise, aerobic exercise, and/or high altitude exercise,and wherein enhancing exercise performance and/or recovery timecomprises reducing exercise-induced increases of plasma inflammatorycytokine levels in the subject or comprises reducing a biomarker ofexercise-induced muscle injury.
 2. (canceled)
 3. The method of claim 2,wherein the exercise-induced plasma inflammatory cytokine is oneselected from the group consisting of interferon-alpha (IFN-alpha),epithelial neutrophil activating protein 78 (ENA-78), and brain-derivedneurotrophic factor (BDNF).
 4. The method of claim 1, wherein enhancingexercise performance comprises at least one of preventing orameliorating exercise-mediated muscle and/or tendon damage and enhancingmuscle and/or tendon recovery therefrom.
 5. The method of claim 4,comprising at least one of preventing or alleviating extent of musclefiber micro-injury, and enhancing recovery therefrom.
 6. The method ofclaim 41, wherein reducing the biomarkers of exercise-induced muscleinjury comprises reducing creatine kinase (CK) and/or, plasma myoglobin.7. The method of claim 4 comprising ameliorating or enhancing recoveringfrom at least one of exercise induced tendinosis, tendonitis,tenosynovitis, avulsion, and tendon strain associated with chronic,repetitive movement.
 8. The method of claim 1, wherein enhancingexercise performance and/or recovery time comprises at least one of:increasing the maximum amount of oxygen that the subject can utilizeduring intense or maximal exercise (VO₂ max); decreasing the rating ofperceived exertion (RPE); reducing exercise-mediated increase bloodlactate levels; preserving muscle contractile function, preservingmaximal force or joint ROM; reducing muscle soreness; and amelioratingthe onset of fatigue in response to the exercise.
 9. The method of claim1, wherein the exercise comprises at least one of intense exercise,eccentric exercise, exercise in elevated ambient temperature, repetitiveexercise, aerobic exercise, and high altitude exercise.
 10. (canceled)11. The method of claim 1, wherein the oxygenated ionic aqueous solutioncomprises oxygen in an amount of at least 25 ppm, at least 30 ppm, atleast 40 ppm, at least 50 ppm, or at least 60 ppm oxygen at ambienttemperature and atmospheric pressure.
 12. The method of claim 1, whereinthe ionic aqueous solution comprises a saline solution.
 13. (canceled)14. The method of claim 1, wherein the administration of the oxygenatedionic aqueous solution comprises oral administration of aqueous solutionor sports beverage.
 15. The method of claim 14, wherein the sportsbeverage comprises a sugar, carbohydrate, electrolyte or other sportsbeverage ingredient.
 16. The method of claim 15, wherein the ionicaqueous solution comprises at least one positively-charged ion selectedfrom the group consisting of: alkali metal based salts; alkali metalbased salts of Li+, Na+, K+, Rb+, and Cs+, alkaline earth based salts;alkaline earth based salts of Mg++ and Ca++; and transition metal-basedpositive ions of Cr, Fe, Co, Ni, Cu, and Zn. 17-31. (canceled)
 32. Themethod of claim 3, wherein the administration of the oxygenated ionicaqueous solution comprises oral administration of aqueous solution orsports beverage.
 33. The method of claim 4, wherein the administrationof the oxygenated ionic aqueous solution comprises oral administrationof aqueous solution or sports beverage.
 34. The method of claim 5,wherein the administration of the oxygenated ionic aqueous solutioncomprises oral administration of aqueous solution or sports beverage.35. The method of claim 8, wherein the administration of the oxygenatedionic aqueous solution comprises oral administration of aqueous solutionor sports beverage.
 36. The method of claim 9, wherein theadministration of the oxygenated ionic aqueous solution comprises oraladministration of aqueous solution or sports beverage.
 37. The method ofclaim 11, wherein the administration of the oxygenated ionic aqueoussolution comprises oral administration of aqueous solution or sportsbeverage.
 38. The method of claim 12, wherein the administration of theoxygenated ionic aqueous solution comprises oral administration ofaqueous solution or sports beverage.